Reagents and methods useful for detecting diseases of the breast

ABSTRACT

A novel member of the uteroglobin family of proteins, designated as BU101, is described. BU101 is defined by a set of contiguous and partially overlapping RNA sequences transcribed from breast tissue, and polypeptides encoded thereby. A fully sequenced clone representing the longest continuous sequence of BU101 is also disclosed. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the breast such as breast cancer. Also provided are antibodies which specifically bind to BU101-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific BU101 polypeptide, which molecules are useful for the therapeutic treatment of breast diseases, tumors or metastases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/697,105, filed Aug. 19, 1996, now abandoned, from which priority isclaimed pursuant to 35 U.S.C. §120 and which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates generally to a novel member of the uteroglobinfamily of proteins that is over-expressed in a percentage of breasttumors. Furthermore, the invention also relates to reagents and methodsfor detecting diseases of the breast. More particularly, the presentinvention relates to reagents such as polynucleotide sequences and thepolypeptide sequences encoded thereby, as well as methods which utilizethese sequences. The polynucleotide and polypeptide sequences are usefulfor detecting, diagnosing, staging, monitoring, prognosticating,preventing or treating, or determining predisposition to diseases orconditions of the breast such as breast cancer.

Breast cancer is the most common form of cancer occurring in females inthe US. The incidence of breast cancers in the United States isprojected to be 180,200 cases diagnosed and 43,900 breast cancer-relateddeaths to occur during 1997 (American Cancer Society statistics).Worldwide, the incidence of breast cancer has increased from 700,000 in1985 to about 900,000 in 1990. G. N. Hortobagyi et al., CA Cancer J Clin45: 199-226 (1995).

Procedures used for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining predispositionto diseases or conditions of the breast, such as breast cancer, are ofcritical importance to the outcome of the patient. For example, patientsdiagnosed with early breast cancer have greater than a 90% five-yearrelative survival rate as compared to a survival rate of about 20% forpatients diagnosed with distantly metastasized breast cancers. (AmericanCancer Society statistics). Currently, the best initial indicators ofearly breast cancer are physical examination of the breast andmammography. J. R. Harris et al. In: Cancer: Principles and Practice ofOncology, Fourth Edition, pp. 1264-1332, Philadelphia, Pa.: J/B.Lippincott Co. (1993). Mammography may detect a breast tumor before itcan be detected by physical examination, but it has limitations. Forexample, the predictive value depends on the observer's skill and thequality of the mammogram. In addition, 80 to 93% of suspiciousmammograms are false positives, and 10 to 15% of women with breastcancer have false negative mammograms. C. J. Wright et al., Lancet 346:29-32 (1995). New diagnostic methods which are more sensitive andspecific for detecting early breast cancer are clearly needed.

Breast cancer patients are closely monitored following initial therapyand during adjuvant therapy to determine response to therapy, and todetect persistent or recurrent disease, or early distant metastasis.Current diagnostic procedures for monitoring breast cancer includemammography, bone scan, chest radiographs, liver function tests andtests for serum markers. The serum tumor markers most commonly used formonitoring patients are carcinoembryonic antigen (CEA) and CA 15-3.Limitations of CEA include absence of elevated serum levels in about 40%of women with metastatic disease. In addition, CEA elevation duringadjuvant therapy may not be related to recurrence but to other factorsthat are not clinically important. CA 15-3 can also be negative in asignificant number of patients with progressive disease and, therefore,fail to predict metastasis. Both CEA and CA 15-3 can be elevated innonmalignant, benign conditions giving rise to false positive results.Therefore, it would be clinically beneficial to find a breast associatedmarker which is more sensitive and specific in detecting cancerrecurrence. J. R. Harris et al., supra. M. K. Schwartz, In: Cancer:Principles and Practice of Oncology. Vol. 1, Fourth Edition, pp.531-542, Philadelphia, Pa.: J/B. Lippincott Co. (1993).

Another important step in managing breast cancer is to determine thestage of the patient's disease because stage determination has potentialprognostic value and provides criteria for designing optimal therapy.Currently, pathological staging of breast cancer is preferable overclinical staging because the former gives a more accurate prognosis. J.R. Harris et al., supra. On the other hand, clinical staging would bepreferred were it at least as accurate as pathological staging, becauseit does not depend on an invasive procedure to obtain tissue forpathological evaluation. Staging of breast cancer could be improved bydetecting new markers in serum or urine which could differentiatebetween different stages of invasion. Such markers could be mRNA orprotein markers expressed by cells originating from the primary tumor inthe breast but residing in blood, bone marrow or lymph nodes and couldserve as sensitive indicators for metastasis to these distal organs. Forexample, specific protein antigens and mRNA, associated with breastepithelial cells, have been detected by immunohistochemical techniquesand RT-PCR, respectively, in bone marrow, lymph nodes and blood ofbreast cancer patients suggesting metastasis. K. Pantel et al.,Onkologie 18: 394-401 (1995).

Such procedures also could include assays based upon the appearance ofvarious disease markers in test samples such as blood, plasma, serum orurine obtained by minimally invasive procedures which are detectable byimmunological methods. These procedures would provide information to aidthe physician in managing the patient with disease of the breast, at lowcost to the patient. Markers such as prostate specific antigen (PSA) andhuman chorionic gonadotropin (hCG) exist and are used clinically forscreening patients for prostate cancer and testicular cancer,respectively. For example, PSA normally is secreted by the prostate athigh levels into the seminal fluid, but is present in very low levels inthe blood of men with normal prostates. Elevated levels of PSA proteinin serum are used in the early detection of prostate cancer or diseasein asymptomatic men. See, for example, G. E. Hanks et al., In: Cancer:Principles and Practice of Oncology, Vol. 1, Fourth Edition, pp.1073-1113, Philadelphia, Pa.: J. B. Lippincott Co. (1993); M. K.Schwartz et al., In: Cancer: Principles and Practice of Oncology, Vol.1, Fourth Edition, pp. 531-542, Philadelphia, Pa.: J. B. Lippincott Co.(1993). Likewise, the management of breast diseases could be improved bythe use of new markers normally expressed in the breast but found inelevated amounts in an inappropriate body compartment as a result of thedisease of the breast. One example is mammaglobin, a member of theuteroglobin family of proteins, which was only detected in breasttissue. Mammaglobin was also found to be over-expressed in some breasttumors versus normal breast tissue using differential display. M. A.Watson et al., Cancer Res. 56:860-865 (1996).

Further, new markers which could predict the biologic behavior of earlybreast cancers would also be of significant value. Early breast cancersthat threaten or will threaten the life of the patient are moreclinically important than those that do not or will not be a threat. G.E. Hanks, supra. Such markers are needed to predict which patients withhistologically negative lymph nodes will experience recurrence of cancerand also to predict which cases of ductal carcinoma in situ will developinto invasive breast carcinoma. More accurate prognostic markers wouldallow the clinician to accurately identify early cancers localized tothe breast which will progress and metastasize if not treatedaggressively. Additionally, the absence of a marker for an aggressivecancer in the patient could spare the patient expensive andnon-beneficial treatment. J. R. Harris et al., supra. E. R. Frykberg etal., Cancer 74: 350-361 (1994).

It would be advantageous, therefore, to provide specific methods andreagents useful for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining predispositionto diseases or conditions of the breast. Such methods would includeassaying a test sample for products of a gene which are overexpressed indiseases and conditions associated with the breast including cancer.Such methods may also include assaying a test sample for products of agene which have been altered by the disease or condition associated withthe breast including cancer. Such methods may further include assaying atest sample for products of a gene whose distribution among the varioustissues and compartments of the body have been altered by abreast-associated disease or condition, including cancer. Such methodswould comprise making cDNA from mRNA in the test sample, amplifying,when necessary, portions of the cDNA corresponding to the gene or afragment thereof, and detecting the cDNA product as an indication of thepresence of the disease or condition, including cancer, or detectingtranslation products of the mRNAs comprising gene sequences as anindication of the presence of the disease. Useful reagents includepolynucleotide(s), or fragment(s) thereof which may be used indiagnostic methods such as reverse transcriptase-polymerase chainreaction (RT-PCR), PCR, or hybridization assays of mRNA extracted frombiopsied tissue, blood or other test samples; or proteins which are thetranslation products of such mRNAs; or antibodies directed against theseproteins. Such assays would include methods for assaying a sample forproduct(s) of the gene and detecting the product(s) as an indication ofdisease of the breast. Drug treatment or gene therapy for diseases andconditions of the breast, including cancer, can be based on theseidentified gene sequences or their expressed proteins, and efficacy ofany particular therapy can be monitored. Furthermore, it would beadvantageous to have available alternative, non-surgical diagnosticmethods capable of detecting early stage breast disease such as cancer.

SUMMARY OF THE INVENTION

The present invention provides a method of detecting a target BU101polynucleotide in a test sample which comprises contacting the testsample with at least one BU101-specific polynucleotide and detecting thepresence of the target BU101 polynucleotide in the test sample. TheBU101-specific polynucleotide has at least 50% identity with apolynucleotide selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof. Also, the BU101 -specific polynucleotide may beattached to a solid phase prior to performing the method.

The present invention also provides a method for detecting BU101 mRNA ina test sample, which comprises performing reverse transcription (RT)with at least one primer in order to produce cDNA, amplifying the cDNAso obtained using BU101 oligonucleotides as sense and antisense primersto obtain BU101 amplicon, and detecting the presence of the BU101amplicon as an indication of the presence of BU101 mRNA in the testsample, wherein the BU101 oligonucleotides have at least 50% identity toa sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof. Amplification can be performed by the polymerasechain reaction. Also, the test sample can be reacted with a solid phaseprior to performing the method, prior to amplification or prior todetection. This reaction can be a direct or an indirect reaction.Further, the detection step can comprise utilizing a detectable labelcapable of generating a measurable signal. The detectable label can beattached to a solid phase.

The present invention further provides a method of detecting a targetBU101 polynucleotide in a test sample suspected of containing targetBU101 polynucleotides, which comprises (a) contacting the test samplewith at least one BU101 oligonucleotide as a sense primer and at leastone BU101 oligonucleotide as an anti-sense primer, and amplifying sameto obtain a first stage reaction product; (b) contacting the first stagereaction product with at least one other BU101 oligonucleotide to obtaina second stage reaction product, with the proviso that the other BU101oligonucleotide is located 3′ to the BU101 oligonucleotides utilized instep (a) and is complementary to the first stage reaction product; and(c) detecting the second stage reaction product as an indication of thepresence of a target BU101 polynucleotide in the test sample. The BU101oligonucleotides selected as reagents in the method have at least 50%identity to a sequence selected from the group consisting of SEQUENCE IDNO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, andfragments or complements thereof. Amplification may be performed by thepolymerase chain reaction. The test sample can be reacted eitherdirectly or indirectly with a solid phase prior to performing themethod, or prior to amplification, or prior to detection. The detectionstep also comprises utilizing a detectable label capable of generating ameasurable signal; further, the detectable label can be attached to asolid phase. Test kits useful for detecting target BU101 polynucleotidesin a test sample are also provided which comprise a container containingat least one BU101-specific polynucleotide selected from the groupconsisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,SEQUENCE ID NO 4, and fragments or complements thereof. These test kitsfurther comprise containers with tools useful for collecting testsamples (such as, for example, blood, urine, saliva and stool). Suchtools include lancets and absorbent paper or cloth for collecting andstabilizing blood; swabs for collecting and stabilizing saliva; and cupsfor collecting and stabilizing urine or stool samples. Collectionmaterials such as, papers, cloths, swabs, cups and the like, mayoptionally be treated to avoid denaturation or irreversible adsorptionof the sample. The collection materials also may be treated with orcontain preservatives, stabilizers or antimicrobial agents to helpmaintain the integrity of the specimens.

The present invention provides a purified polynucleotide or fragmentthereof derived from a BU101 gene. The purified polynucleotide iscapable of selectively hybridizing to the nucleic acid of the BU101gene, or a complement thereof. The polynucleotide has at least 60%identity to a polynucleotide selected from the group consisting ofSEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4,and fragments or complements thereof. Further, the purifiedpolynucleotide can be produced by recombinant and/or synthetictechniques. The purified recombinant polynucleotide can be containedwithin a recombinant vector. The invention further comprises a host celltransfected with said vector.

The present invention further provides a recombinant expression systemcomprising a nucleic acid sequence that includes an open reading framederived from BU101. The nucleic acid sequence has at least 50% identitywith a sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof. The nucleic acid sequence is operably linked to acontrol sequence compatible with a desired host. Also provided is a celltransfected with this recombinant expression system.

The present invention also provides polypeptides encoded by BU101. Thepolypeptides can be produced by recombinant technology, provided inpurified form, or produced by synthetic techniques. The polypeptidescomprise amino acid sequences which have at least 60% identity to anamino acid sequence selected from the group consisting of SEQUENCE IDNOS 15-23, or at least 90% identity with the sequence of a fragment ofSEQUENCE ID NO 15.

Also provided is an antibody which specifically binds to at least oneBU101 epitope. The antibody can be a polyclonal or monoclonal antibody.The epitope is derived from an amino acid sequence selected from thegroup consisting of SEQUENCE ID NOS 15-23, and fragments thereof. Assaykits for determining the presence of BU101 antigen or anti-BU101antibody in a test sample are also included. In one embodiment, theassay kits comprise a container containing at least one BU101polypeptide having at least 50% identity to an amino acid sequenceselected from the group consisting of SEQUENCE ID NOS 15-23, andfragments thereof. Further, the test kit can comprise a container withtools useful for collecting test samples (such as blood, urine, salivaand stool). Such tools include lancets and absorbent paper or cloth forcollecting and stabilizing blood; swabs for collecting and stabilizingsaliva; and cups for collecting and stabilizing urine or stool samples.Collection materials such as, papers, cloths, swabs, cups and the like,may optionally be treated to avoid denaturation or irreversibleadsorption of the sample. These collection materials also may be treatedwith or contain preservatives, stabilizers or antimicrobial agents tohelp maintain the integrity of the specimens. Also, the polypeptide canbe attached to a solid phase.

Another assay kit for determining the presence of BU101 antigen oranti-BU101 antibody in a test sample comprises a container containing anantibody which specifically binds to a BU101 antigen, wherein the BU101antigen comprises at least one BU101-encoded epitope. The BU101 antigenhas at least about 60% sequence similarity to a sequence of aBU101-encoded antigen selected from the group consisting of SEQUENCE IDNOS 15-23, and fragments thereof. These test kits can further comprisecontainers with tools useful for collecting test samples (such as blood,urine, saliva and stool). Such tools include lancets and absorbent paperor cloth for collecting and stabilizing blood; swabs for collecting andstabilizing saliva; cups for collecting and stabilizing urine or stoolsamples. Collection materials, papers, cloths, swabs, cups and the like,may optionally be treated to avoid denaturation or irreversibleadsorption of the sample. These collection materials also may be treatedwith, or contain, preservatives, stabilizers or antimicrobial agents tohelp maintain the integrity of the specimens. The antibody can beattached to a solid phase.

A method for producing a polypeptide which contains at least one epitopeof BU101 is provided, which method comprises incubating host cellstransfected with an expression vector. This vector comprises apolynucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence having at least 50% identity to a BU101amino acid sequence selected from the group consisting of SEQUENCE IDNOS 15-23, and fragments thereof.

A method for detecting BU101 antigen in a test sample suspected ofcontaining BU101 antigen also is provided. The method comprisescontacting the test sample with an antibody or fragment thereof whichspecifically binds to at least one epitope of a BU101 antigen, for atime and under conditions sufficient for the formation ofantibody/antigen complexes; and detecting the presence of such complexescontaining the antibody as an indication of the presence of BU101antigen in the test sample. The antibody can be attached to a solidphase and be either a monoclonal or polyclonal antibody. Furthermore,the antibody specifically binds to at least one BU101 antigen selectedfrom the group consisting of SEQUENCE ID NOS 15-23, and fragmentsthereof.

Another method is provided which detects antibodies which specificallybind to BU101 antigen in a test sample suspected of containing theseantibodies. The method comprises contacting the test sample with apolypeptide which contains at least one BU101 epitope, wherein the BU101epitope comprises an amino acid sequence having at least 50% identitywith an amino acid sequence encoded by a BU101 polynucleotide, or afragment thereof. Contacting is carried out for a time and underconditions sufficient to allow antigen/antibody complexes to form. Themethod further entails detecting complexes which contain thepolypeptide. The polypeptide can be attached to a solid phase. Further,the polypeptide can be a recombinant protein or a synthetic peptidehaving at least 50% identity to an amino acid sequence selected from thegroup consisting of SEQUENCE ID NOS 15-23, and fragments thereof.

The present invention provides a cell transfected with a BU101 nucleicacid sequence that encodes at least one epitope of a BU101 antigen, orfragment thereof. The nucleic acid sequence is selected from the groupconsisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,SEQUENCE ID NO 4, and fragments or complements thereof.

A method for producing antibodies to BU101 antigen also is provided,which method comprises administering to an individual an isolatedimmunogenic polypeptide or fragment thereof, wherein the isolatedimmunogenic polypeptide comprises at least one BU101 epitope in anamount sufficient to produce an immune response. The isolated,immunogenic polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQUENCE ID NOS 15-23, and fragments thereof.

Another method for producing antibodies which specifically bind to BU101antigen is disclosed, which method comprises administering to a mammal aplasmid comprising a nucleic acid sequence which encodes at least oneBU101 epitope derived from an amino acid sequence selected from thegroup consisting of SEQUENCE ID NOS 15-23, and fragments thereof.

Also provided is a composition of matter that comprises a BU101polynucleotide of at least about 10-12 nucleotides having at least 60%identity to a polynucleotide selected from the group consisting ofSEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4,and fragments or complements thereof. The BU101 polynucleotide encodesan amino acid sequence having at least one BU101 epitope. Anothercomposition of matter provided by the present invention comprises apolypeptide with at least one BU101 epitope of about 8-10 amino acids.The polypeptide comprises an amino acid sequence having at least 60%identity to an amino acid sequence selected from the group consisting ofSEQUENCE ID NOS 15-23, or at least 90% identity with the sequence of afragment of SEQUENCE ID NO 15. Also provided is a gene or fragmentthereof coding for a BU101 polypeptide which has at least 60% identityto SEQUENCE ID NO 15, and a gene or a fragment thereof comprising DNAhaving at least 60% identity to SEQUENCE ID NO 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide alignment of clones 603148 (SEQUENCE ID NO1), 604290 (SEQUENCE ID NO 2), and the consensus sequence (SEQUENCE IDNO 3) derived therefrom;

FIG. 2 shows the contig map depicting the formation of the consensusnucleotide sequence (SEQUENCE ID NO 3) from the nucleotide alignment ofoverlapping clones 603148 (SEQUENCE ID NO 1) and 604290 (SEQUENCE ID NO2);

FIG. 3A represents a scan of an ethidium bromide stained agarose gel ofRNA from various tissue extracts;

FIG. 3B shows a northern blot of RNA from various tissue extracts usingBU101 radiolabeled probe;

FIG. 4 represents a scan of a stained agarose gel of BU101-specificprimed PCR amplification products;

FIG. 5 is a representation of a film of a western blot of differenttissue protein extracts using BU101.8 antiserum.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a gene or a fragment thereof which codesfor a BU101 polypeptide having at least about 60% identity to SEQUENCEID NO 15. The present invention further encompasses a BU101 gene or afragment thereof comprising DNA which has at least about 60% identity toSEQUENCE ID NO 4.

The present invention provides methods for assaying a test sample forproducts of a breast tissue gene designated as BU101, which comprisesmaking cDNA from mRNA in the test sample, and detecting the cDNA as anindication of the presence of breast tissue gene BU101. The method mayinclude an amplification step, wherein one or more portions of the mRNAfrom BU101 corresponding to the gene or fragments thereof, is amplified.Methods also are provided for assaying for the translation products ofBU101. Test samples which may be assayed by the methods provided hereininclude tissues, cells, body fluids and secretions. The presentinvention also provides reagents such as oligonucleotide primers andpolypeptides which are useful in performing these methods.

Portions of the nucleic acid sequences disclosed herein are useful asprimers for the reverse transcription of RNA or for the amplification ofcDNA; or as probes to determine the presence of certain mRNA sequencesin test samples. Also disclosed are nucleic acid sequences which permitthe production of encoded polypeptide sequences which are useful asstandards or reagents in diagnostic immunoassays, as targets forpharmaceutical screening assays and/or as components or as target sitesfor various therapies. Monoclonal and polyclonal antibodies directedagainst at least one epitope contained within these polypeptidesequences are useful as delivery agents for therapeutic agents as wellas for diagnostic tests and for screening for diseases or conditionsassociated with BU101, especially breast cancer. Isolation of sequencesof other portions of the gene of interest can be accomplished utilizingprobes or PCR primers derived from these nucleic acid sequences. Thisallows additional probes of the mRNA or cDNA of interest to beestablished, as well as corresponding encoded polypeptide sequences.These additional molecules are useful in detecting, diagnosing, staging,monitoring, prognosticating, preventing or treating, or determining thepredisposition to, diseases and conditions of the breast such as breastcancer, characterized by BU101, as disclosed herein.

Techniques for determining amino acid sequence “similarity” arewell-known in the art. In general, “similarity” means the exact aminoacid to amino acid comparison of two or more polypeptides at theappropriate place, where amino acids are identical or possess similarchemical and/or physical properties such as charge or hydrophobicity. Aso-termed “percent similarity” then can be determined between thecompared polypeptide sequences. Techniques for determining nucleic acidand amino acid sequence identity also are well known in the art andinclude determining the nucleotide sequence of the mRNA for that gene(usually via a cDNA intermediate) and determining the amino acidsequence encoded thereby, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity.” Two or more amino acid sequences likewise can be compared bydetermining their “percent identity.” The programs available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, arecapable of calculating both the identity between two polynucleotides andthe identity and similarity between two polypeptide sequences,respectively. Other programs for calculating identity or similaritybetween sequences are known in the art.

The compositions and methods described herein will enable theidentification of certain markers as indicative of a breast tissuedisease or condition; the information obtained therefrom will aid in thedetecting, diagnosing, staging, monitoring, prognosticating, preventingor treating, or determining diseases or conditions associated withBU101, especially breast cancer. Test methods include, for example,probe assays which utilize the sequence(s) provided herein and whichalso may utilize nucleic acid amplification methods such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), andhybridization. In addition, the nucleotide sequences provided hereincontain open reading frames from which an immunogenic epitope may befound. This epitope is believed to be unique to the disease state orcondition associated with BU101. It also is thought that thepolynucleotides or polypeptides and protein encoded by the BU101 geneare useful as a marker. This marker is either elevated in disease suchas breast cancer, altered in disease such as breast cancer, or presentas a normal protein but appearing in an inappropriate body compartment.The uniqueness of the epitope may be determined by (i) its immunologicalreactivity and specificity with antibodies directed against proteins andpolypeptides encoded by the BU101 gene, and (ii) its nonreactivity withany other tissue markers. Methods for determining immunologicalreactivity are well-known and include but are not limited to, forexample, radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), hemagglutination (HA), fluorescence polarization immunoassay(FPIA), chemiluminescent immunoassay (CLIA) and others. Several examplesof suitable methods are described herein.

Unless otherwise stated, the following terms shall have the followingmeanings:

A polynucleotide “derived from” or “specific for” a designated sequencerefers to a polynucleotide sequence which comprises a contiguoussequence of approximately at least about 6 nucleotides, preferably atleast about 8 nucleotides, more preferably at least about 10-12nucleotides, and even more preferably at least about 15-20 nucleotidescorresponding, i.e., identical or complementary to, a region of thedesignated nucleotide sequence. The sequence may be complementary oridentical to a sequence which is unique to a particular polynucleotidesequence as determined by techniques known in the art. Comparisons tosequences in databanks, for example, can be used as a method todetermine the uniqueness of a designated sequence. Regions from whichsequences may be derived, include but are not limited to, regionsencoding specific epitopes, as well as non-translated and/ornon-transcribed regions.

The derived polynucleotide will not necessarily be derived physicallyfrom the nucleotide sequence of interest under study, but may begenerated in any manner, including but not limited to chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide. In addition, combinations of regions corresponding tothat of the designated sequence may be modified in ways known in the artto be consistent with the intended use.

A “fragment” of a specified polynucleotide refers to a polynucleotidesequence which comprises a contiguous sequence of approximately at leastabout 6 nucleotides, preferably at least about 8 nucleotides, morepreferably at least about 10-12 nucleotides, and even more preferably atleast about 15-20 nucleotides corresponding, i.e., identical orcomplementary to, a region of the specified nucleotide sequence.

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by either DNA polymerase, RNApolymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g., PNA as defined hereinbelow) which can be used toidentify a specific polynucleotide present in samples bearing thecomplementary sequence.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.Thus, a “polypeptide,” “protein,” or “amino acid” sequence has at leastabout 50% identity, preferably about 60% identity, more preferably about75-85% identity, and most preferably about 90-95% or more identity to aBU101 amino acid sequence. Further, the BU101 “polypeptide,” “protein,”or “amino acid” sequence may have at least about 60% similarity,preferably at least about 75% similarity, more preferably about 85%similarity, and most preferably about 95% or more similarity to apolypeptide or amino acid sequence of BU101. This amino acid sequencecan be selected from the group consisting of SEQUENCE ID NOS 15-23, andfragments thereof.

A “recombinant polypeptide,” “recombinant protein,” or “a polypeptideproduced by recombinant techniques,” which terms may be usedinterchangeably herein, describes a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system.

The term “synthetic peptide” as used herein means a polymeric form ofamino acids of any length, which may be chemically synthesized bymethods well-known to the routineer. These synthetic peptides are usefulin various applications.

The term “polynucleotide” as used herein means a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA,as well as double- and single-stranded RNA. It also includesmodifications, such as methylation or capping and unmodified forms ofthe polynucleotide. The terms “polynucleotide,” “oligomer,”“oligonucleotide,” and “oligo” are used interchangeably herein.

“A sequence corresponding to a cDNA” means that the sequence contains apolynucleotide sequence that is identical or complementary to a sequencein the designated DNA. The degree (or “percent”) of identity orcomplementarity to the cDNA will be approximately 50% or greater,preferably at least about 60-70% or greater, and more preferably atleast about 90%. or greater. The sequence that corresponds to theidentified cDNA will be at least about 50 nucleotides in length,preferably at least about 60 nucleotides in length, and more preferablyat least about 70 nucleotides in length. The correspondence between thegene or gene fragment of interest and the cDNA can be determined bymethods known in the art and include, for example, a direct comparisonof the sequenced material with the cDNAs described, or hybridization anddigestion with single strand nucleases, followed by size determinationof the digested fragments.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

“Purified polypeptide” or “purified protein” means a polypeptide ofinterest or fragment thereof which is essentially free of, e.g.,contains less than about 50%, preferably less than about 70%, and morepreferably less than about 90%, cellular components with which thepolypeptide of interest is naturally associated. Methods for purifyingpolypeptides of interest are known in the art.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

“Polypeptide” and “protein” are used interchangeably herein and indicateat least one molecular chain of amino acids linked through covalentand/or non-covalent bonds. The terms do not refer to a specific lengthof the product. Thus peptides, oligopeptides and proteins are includedwithin the definition of polypeptide. The terms includepost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Inaddition, protein fragments, analogs, mutated or variant proteins,fusion proteins and the like are included within the meaning ofpolypeptide.

A “fragment” of a specified polypeptide refers to an amino acid sequencewhich comprises at least about 5 amino acids, more preferably at leastabout 8-10 amino acids, and even more preferably at least about 15-20amino acids derived from the specified polypeptide.

“Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cellcultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vectorsor other transferred DNA, and include the original progeny of theoriginal cell which has been transfected.

As used herein “replicon” means any genetic element, such as a plasmid,a chromosome or a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell.

A “vector” is a replicon in which another polynucleotide segment isattached, such as to bring about the replication and/or expression ofthe attached segment.

The term “control sequence” refers to a polynucleotide sequence which isnecessary to effect the expression of a coding sequence to which it isligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, such control sequences generally includea promoter, a ribosomal binding site, and terminators; in eukaryotes,such control sequences generally include promoters, terminators and, insome instances, enhancers. The term “control sequence” thus is intendedto include at a minimum all components whose presence is necessary forexpression, and also may include additional components whose presence isadvantageous, for example, leader sequences.

“Operably linked” refers to a situation wherein the components describedare in a relationship permitting them to function in their intendedmanner. Thus, for example, a control sequence “operably linked” to acoding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequence.

The term “open reading frame” or “ORF” refers to a region of apolynucleotide sequence which encodes a polypeptide. This region mayrepresent a portion of a coding sequence or a total coding sequence.

A “coding sequence” is a polynucleotide sequence which is transcribedinto mRNA and translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to, mRNA, cDNA and recombinantpolynucleotide sequences.

The term “immunologically identifiable with/as” refers to the presenceof epitope(s) and polypeptide(s) which also are present in and areunique to the designated polypeptide(s). Immunological identity may bedetermined by antibody binding and/or competition in binding. Thesetechniques are known to the routineer and also are described herein. Theuniqueness of an epitope also can be determined by computer searches ofknown data banks, such as GenBank, for the polynucleotide sequence whichencodes the epitope and by amino acid sequence comparisons with otherknown proteins.

As used herein, “epitope” means an antigenic determinant of apolypeptide or protein. Conceivably, an epitope can comprise three aminoacids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids andmore usually, it consists of at least eight to ten amino acids. Methodsof examining spatial conformation are known in the art and include, forexample, x-ray crystallography and two-dimensional nuclear magneticresonance.

A “conformational epitope” is an epitope that is comprised of specificjuxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

A polypeptide is “immunologically reactive” with an antibody when itbinds to an antibody due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly, by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

As used herein, the term “immunogenic polypeptide containing an epitopeof interest” means naturally occurring polypeptides of interest orfragments thereof, as well as polypeptides prepared by other means, forexample, by chemical synthesis or the expression of the polypeptide in arecombinant organism.

The term “transfection” refers to the introduction of an exogenouspolynucleotide into a prokaryotic or eucaryotic host cell, irrespectiveof the method used for the introduction. The term “transfection” refersto both stable and transient introduction of the polynucleotide, andencompasses direct uptake of polynucleotides, transformation,transduction, and f-mating. Once introduced into the host cell, theexogenous polynucleotide may be maintained as a non-integrated replicon,for example, a plasmid, or alternatively, may be integrated into thehost genome.

“Treatment” refers to prophylaxis and/or therapy.

The term “individual” as used herein refers to vertebrates, particularlymembers of the mammalian species and includes, but is not limited to,domestic animals, sports animals, primates and humans; more particularlythe term refers to humans.

The term “sense strand” or “plus strand” (or “+”) as used herein denotesa nucleic acid that contains the sequence that encodes the polypeptide.The term “antisense strand” or “minus strand” (or “−”) denotes a nucleicacid that contains a sequence that is complementary to that of the“plus” strand.

The term “test sample” refers to a component of an individual's bodywhich is the source of the analyte (such as, antibodies of interest orantigens of interest). These components are well known in the art. Atest sample is typically anything suspected of containing a targetsequence. Test samples can be prepared using methodologies well known inthe art such as by obtaining a specimen from an individual and, ifnecessary, disrupting any cells contained thereby to release targetnucleic acids. These test samples include biological samples which canbe tested by the methods of the present invention described herein andinclude human and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates,urine, lymph fluids and various external secretions of the respiratory,intestinal and genitourinary tracts, tears, saliva, milk, white bloodcells, myelomas and the like; biological fluids such as cell culturesupernatants; tissue specimens which may be fixed; and cell specimenswhich may be fixed.

“Purified product” refers to a preparation of the product which has beenisolated from the cellular constituents with which the product isnormally associated and from other types of cells which may be presentin the sample of interest.

“PNA” denotes a “peptide nucleic acid analog” which may be utilized in aprocedure such as an assay described herein to determine the presence ofa target. “MA” denotes a “morpholino analog” which may be utilized in aprocedure such as an assay described herein to determine the presence ofa target. See, for example, U.S. Pat. No. 5,378,841, which isincorporated herein by reference. PNAs are neutrally charged moietieswhich can be directed against RNA targets or DNA. PNA probes used inassays in place of, for example, the DNA probes of the presentinvention, offer advantages not achievable when DNA probes are used.These advantages include manufacturability, large scale labeling,reproducibility, stability, insensitivity to changes in ionic strengthand resistance to enzymatic degradation which is present in methodsutilizing DNA or RNA. These PNAs can be labeled with (“attached to”)such signal generating compounds as fluorescein, radionucleotides,chemiluminescent compounds and the like. PNAs or other nucleic acidanalogs such as MAs thus can be used in assay methods in place of DNA orRNA. Although assays are described herein utilizing DNA probes, it iswithin the scope of the routineer that PNAs or MAs can be substitutedfor RNA or DNA with appropriate changes if and as needed in assayreagents.

“Analyte,” as used herein, is the substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as, anantibody), or for which a specific binding member can be prepared. Thus,an analyte is a substance that can bind to one or more specific bindingmembers in an assay. “Analyte” also includes any antigenic substances,haptens, antibodies and combinations thereof. As a member of a specificbinding pair, the analyte can be detected by means of naturallyoccurring specific binding partners (pairs) such as the use of intrinsicfactor protein as a member of a specific binding pair for thedetermination of Vitamin B12, the use of folate-binding protein todetermine folic acid, or the use of a lectin as a member of a specificbinding pair for the determination of a carbohydrate. The analyte caninclude a protein, a polypeptide, an amino acid, a nucleotide target andthe like.

“Diseases of the breast” or “breast disease,” or “condition of thebreast” as used herein, refer to any disease or condition of the breastincluding, but not limited to, atypical hyperplasia, fibroadenoma,cystic breast disease, and cancer.

“Breast cancer,” as used herein, refers to any malignant disease of thebreast including, but not limited to, ductal carcinoma in situ, lobularcarcinoma in situ, infiltrating ductal carcinoma, medullary carcinoma,tubular carcinoma, mucinous carcinoma, infiltrating lobular carcinoma,infiltrating comedocarcinoma and inflammatory carcinoma.

An “Expressed Sequence Tag” or “EST” refers to the partial sequence of acDNA insert which has been made by reverse transcription of mRNAextracted from a tissue followed by insertion into a vector.

A “transcript image” refers to a table or list giving the quantitativedistribution of ESTs in a library and represents the genes active in thetissue from which the library was made.

The present invention provides assays which utilize specific bindingmembers. A “specific binding member,” as used herein, is a member of aspecific binding pair. That is, two different molecules where one of themolecules, through chemical or physical means, specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, antibodies and antibody fragments, both monoclonal andpolyclonal and complexes thereof, including those formed by recombinantDNA molecules.

The term “hapten,” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

A “capture reagent,” as used herein, refers to an unlabeled specificbinding member which is specific either for the analyte as in a sandwichassay, for the indicator reagent or analyte as in a competitive assay,or for an ancillary specific binding member, which itself is specificfor the analyte, as in an indirect assay. The capture reagent can bedirectly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

“Specific binding member” as used herein means a member of a specificbinding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule.

The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means, conjugated (“attached”) to aspecific binding member. In addition to being an antibody member of aspecific binding pair, the indicator reagent also can be a member of anyspecific binding pair, including either hapten-anti-hapten systems suchas biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin,a complementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to the polypeptide of interest as in a sandwich assay, to thecapture reagent as in a competitive assay, or to the ancillary specificbinding member as in an indirect assay. When describing probes and probeassays, the term “reporter molecule” may be used. A reporter moleculecomprises a signal generating compound as described hereinaboveconjugated to a specific binding member of a specific binding pair, suchas carbazole or adamantane.

The various “signal-generating compounds” (labels) contemplated includechromagens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase and the like.The selection of a particular label is not critical, but it must becapable of producing a signal either by itself or in conjunction withone or more additional substances.

“Solid phases” (“solid supports”) are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic or non-magnetic beads, nitrocellulose strips, membranes,microparticles such as latex particles, sheep (or other animal) redblood cells and Duracytes® (red blood cells “fixed” by pyruvic aldehydeand formaldehyde, available from Abbott Laboratories, Abbott Park, Ill.)and others. The “solid phase” is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and Duracytes® are all suitable examples. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like. A “solid phase,” as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid phase and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, sheep (orother suitable animal's) red blood cells, Duracytes® and otherconfigurations known to those of ordinary skill in the art.

It is contemplated and within the scope of the present invention thatthe solid phase also can comprise any suitable porous material withsufficient porosity to allow access by detection antibodies and asuitable surface affinity to bind antigens. Microporous structuresgenerally are preferred, but materials with a gel structure in thehydrated state may be used as well. Such useful solid supports include,but are not limited to, nitrocellulose and nylon. It is contemplatedthat such porous solid supports described herein preferably are in theform of sheets of thickness from about 0.01 to 0.5 mm, preferably about0.1 mm. The pore size may vary within wide limits and preferably is fromabout 0.025 to 15 microns, especially from about 0.15 to 15 microns. Thesurface of such supports may be activated by chemical processes whichcause covalent linkage of the antigen or antibody to the support. Theirreversible binding of the antigen or antibody is obtained, however, ingeneral, by adsorption on the porous material by poorly understoodhydrophobic forces. Other suitable solid supports are known in the art.

Reagents

The present invention provides reagents such as polynucleotide sequencesderived from a breast tissue of interest and designated as BU101,polypeptides encoded thereby and antibodies specific for thesepolypeptides. The present invention also provides reagents such asoligonucleotide fragments derived from the disclosed polynucleotides andnucleic acid sequences complementary to these polynucleotides. Thepolynucleotides , polypeptides, or antibodies of the present inventionmay be used to provide information leading to the detecting, diagnosing,staging, monitoring, prognosticating, preventing or treating of, ordetermining the predisposition to, diseases and conditions of the breastsuch as cancer. The sequences disclosed herein represent uniquepolynucleotides which can be used in assays or for producing a specificprofile of gene transcription activity. Such assays are disclosed inEuropean Patent Number 0373203B1 and International Publication No. WO95/11995, which are hereby incorporated by reference.

Selected BU101-derived polynucleotides can be used in the methodsdescribed herein for the detection of normal or altered gene expression.Such methods may employ BU101 polynucleotides or oligonucleotides,fragments or derivatives thereof, or nucleic acid sequencescomplementary thereto.

The polynucleotides disclosed herein, their complementary sequences, orfragments of either, can be used in assays to detect, amplify orquantify genes, nucleic acids, cDNAs or mRNAs relating to breast tissuedisease and conditions associated therewith. They also can be used toidentify an entire or partial coding region of a BU101 polypeptide. Theyfurther can be provided in individual containers in the form of a kitfor assays, or provided as individual compositions. If provided in a kitfor assays, other suitable reagents such as buffers, conjugates and thelike may be included.

The polynucleotide may be in the form of RNA or DNA. Polynucleotides inthe form of DNA, cDNA, genomic DNA, nucleic acid analogs and syntheticDNA are within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded, may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence which encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as the DNA provided herein.

This polynucleotide may include only the coding sequence for thepolypeptide, or the coding sequence for the polypeptide and anadditional coding sequence such as a leader or secretory sequence or aproprotein sequence, or the coding sequence for the polypeptide (andoptionally an additional coding sequence) and a non-coding sequence,such as a non-coding sequence 5′ and/or 3′ of the coding sequence forthe polypeptide.

In addition, the invention includes variant polynucleotides containingmodifications such as polynucleotide deletions, substitutions oradditions; and any polypeptide modification resulting from the variantpolynucleotide sequence. A polynucleotide of the present invention alsomay have a coding sequence which is a naturally occurring allelicvariant of the coding sequence provided herein.

In addition, the coding sequence for the polypeptide may be fused in thesame reading frame to a polynucleotide sequence which aids in expressionand secretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the polypeptide. The polynucleotides may alsoencode for a proprotein which is the protein plus additional 5′ aminoacid residues. A protein having a prosequence is a proprotein and may,in some cases, be an inactive form of the protein. Once the prosequenceis cleaved an active protein remains. Thus, the polynucleotide of thepresent invention may encode for a protein, or for a protein having aprosequence, or for a protein having both a presequence (leadersequence) and a prosequence.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. a COS-7 cell line, isused. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein. See, for example, I. Wilson et al., Cell 37:767(1984).

It is contemplated that polynucleotides will be considered to hybridizeto the sequences provided herein if there is at least 50%, preferably atleast 70%, and more preferably at least 90% identity between thepolynucleotide and the sequence.

The present invention also provides an antibody produced by using apurified BU101 polypeptide of which at least a portion of thepolypeptide is encoded by a BU101 polynucleotide selected from thepolynucleotides provided herein. These antibodies may be used in themethods provided herein for the detection of BU101 antigen in testsamples. The presence of BU101 antigen in the test samples is indicativeof the presence of a breast disease or condition. The antibody also maybe used for therapeutic purposes, for example, in neutralizing theactivity of BU101 polypeptide in conditions associated with altered orabnormal expression.

The present invention further relates to a BU101 polypeptide which hasthe deduced amino acid sequence as provided herein, as well asfragments, analogs and derivatives of such polypeptide. The polypeptideof the present invention may be a recombinant polypeptide, a naturalpurified polypeptide or a synthetic polypeptide. The fragment,derivative or analog of the BU101 polypeptide may be one in which one ormore of the amino acid residues is substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or it may be one in which one or more ofthe amino acid residues includes a substituent group; or it may be onein which the polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or it may be one in which the additional aminoacids are fused to the polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are within the scope of the present invention. The polypeptidesand polynucleotides of the present invention are provided preferably inan isolated form and preferably purified.

Thus, a polypeptide of the present invention may have an amino acidsequence that is identical to that of the naturally occurringpolypeptide or that is different by minor variations due to one or moreamino acid substitutions. The variation may be a “conservative change”typically in the range of about 1 to 5 amino acids, wherein thesubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine or threonine with serine.In contrast, variations may include nonconservative changes, e.g.,replacement of a glycine with a tryptophan. Similar minor variations mayalso include amino acid deletions or insertions, or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without changing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software (DNASTAR Inc., Madison Wis.).

Probes constructed according to the polynucleotide sequences of thepresent invention can be used in various assay methods to providevarious types of analysis.

For example, such probes can be used in fluorescent in situhybridization (FISH) technology to perform chromosomal analysis, andused to identify cancer-specific structural alterations in thechromosomes, such as deletions or translocations that are visible fromchromosome spreads or detectable using PCR-generated and/or allelespecific oligonucleotides probes, allele specific amplification or bydirect sequencing. Probes also can be labeled with radioisotopes,directly- or indirectly-detectable haptens, or fluorescent molecules,and utilized for in situ hybridization studies to evaluate the MRNAexpression of the gene comprising the polynucleotide in tissue specimensor cells.

This invention also provides teachings as to the production of thepolynucleotides and polypeptides provided herein.

Probe Assays

The sequences provided herein may be used to produce probes which can beused in assays for the detection of nucleic acids in test samples. Theprobes may be designed from conserved nucleotide regions of thepolynucleotides of interest or from non-conserved nucleotide regions ofthe polynucleotide of interest. The design of such probes foroptimization in assays is within the skill of the routineer. Generally,nucleic acid probes are developed from non-conserved or unique regionswhen maximum specificity is desired, and nucleic acid probes aredeveloped from conserved regions when assaying for nucleotide regionsthat are closely related to, for example, different members of amulti-gene family or in related species like mouse and man.

The polymerase chain reaction (PCR) is a technique for amplifying adesired nucleic acid sequence (target) contained in a nucleic acid ormixture thereof. In PCR, a pair of primers are employed in excess tohybridize to the complementary strands of the target nucleic acid. Theprimers are each extended by a polymerase using the target nucleic acidas a template. The extension products become target sequencesthemselves, following dissociation from the original target strand. Newprimers then are hybridized and extended by a polymerase, and the cycleis repeated to geometrically increase the number of target sequencemolecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202,which are incorporated herein by reference.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acidamplification. In LCR, probe pairs are used which include two primary(first and second) and two secondary (third and fourth) probes, all ofwhich are employed in molar excess to target. The first probe hybridizesto a first segment of the target strand, and the second probe hybridizesto a second segment of the target strand, the first and second segmentsbeing contiguous so that the primary probes abut one another in 5′phosphate-3′ hydroxyl relationship, and so that a ligase can covalentlyfuse or ligate the two probes into a fused product. In addition, a third(secondary) probe can hybridize to a portion of the first probe and afourth (secondary) probe can hybridize to a portion of the second probein a similar abutting fashion. Of course, if the target is initiallydouble stranded, the secondary probes also will hybridize to the targetcomplement in the first instance. Once the ligated strand of primaryprobes is separated from the target strand, it will hybridize with thethird and fourth probes which can be ligated to form a complementary,secondary ligated product. It is important to realize that the ligatedproducts are functionally equivalent to either the target or itscomplement. By repeated cycles of hybridization and ligation,amplification of the target sequence is achieved. This technique isdescribed more completely in EP-A-320 308 to K. Backman published Jun.16, 1989 and EP-A-439 182 to K. Backman et al, published Jul. 31, 1991,both of which are incorporated herein by reference.

For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770, which is incorporated herein byreference; or reverse transcribe mRNA into cDNA followed by asymmetricgap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall etal., PCR Methods and Applications 4: 80-84 (1994), which also isincorporated herein by reference.

Other known amplification methods which can be utilized herein includebut are not limited to the so-called “NASBA” or “3SR” techniquedescribed by J. C. Guatelli, et al., PNAS USA 87:1874-1878 (1990) andalso described by J. Compton, Nature 350 (No. 6313):91-92 (1991); Q-betaamplification as described in published European Patent Application(EPA) No. 4544610; strand displacement amplification (as described in G.T. Walker et al., Clin. Chem. 42:9-13 (1996)) and European PatentApplication No. 684315; and target-mediated amplification, as describedin International Publication No. WO 93/22461.

Detection of BU101 may be accomplished using any suitable detectionmethod, including those detection methods which are currently well knownin the art, as well as detection strategies which may evolve later.Examples of the foregoing presently known detection methods are herebyincorporated herein by reference. See, for example, Caskey et al., U.S.Pat. No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015. Examples ofsuch detection methods include target amplification methods as well assignal amplification technologies. An example of presently knowndetection methods would include the nucleic acid amplificationtechnologies referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, forexample, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand et al., U.S.Pat. No. 5,210,015. All of the foregoing are hereby incorporated byreference. Detection may also be accomplished using signal amplificationsuch as that disclosed in Snitman et al., U.S. Pat. No. 5,273,882. Whilethe amplification of target or signal is preferred at present, it iscontemplated and within the scope of the present invention thatultrasensitive detection methods which do not require amplification canbe utilized herein.

Detection, both amplified and non-amplified, may be (combined) carriedout using a variety of heterogeneous and homogeneous detection formats.Examples of heterogeneous detection formats are disclosed in Snitman etal., U.S. Pat. No. 5,273,882, Albarella et al in EP-84114441.9, Urdea etal., U.S. Pat. No. 5,124,246, Ullman et al. U.S. Pat. No. 5,185,243 andKourilsky et al., U.S. Pat. No. 4,581,333. All of the foregoing arehereby incorporated by reference. Examples of homogeneous detectionformats are disclosed in, Caskey et al., U.S. Pat. No. 5,582,989,Gelfand et al., U.S. Pat. No. 5,210,015, which are incorporated hereinby reference. Also contemplated and within the scope of the presentinvention is the use of multiple probes in the hybridization assay,which use improves sensitivity and amplification of the BU101 signal.See, for example, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand etal., U.S. Pat. No. 5,210,015, which are incorporated herein byreference.

In one embodiment, the present invention generally comprises the stepsof contacting a test sample suspected of containing a targetpolynucleotide sequence with amplification reaction reagents comprisingan amplification primer, and a detection probe that can hybridize withan internal region of the amplicon sequences. Probes and primersemployed according to the method provided herein are labeled withcapture and detection labels, wherein probes are labeled with one typeof label and primers are labeled with another type of label.Additionally, the primers and probes are selected such that the probesequence has a lower melt temperature than the primer sequences. Theamplification reagents, detection reagents and test sample are placedunder amplification conditions whereby, in the presence of targetsequence, copies of the target sequence (an amplicon) are produced. Inthe usual case, the amplicon is double stranded because primers areprovided to amplify a target sequence and its complementary strand. Thedouble stranded amplicon then is thermally denatured to produce singlestranded amplicon members. Upon formation of the single strandedamplicon members, the mixture is cooled to allow the formation ofcomplexes between the probes and single stranded amplicon members.

As the single stranded amplicon sequences and probe sequences arecooled, the probe sequences preferentially bind the single strandedamplicon members. This finding is counterintuitive given that the probesequences generally are selected to be shorter than the primer sequencesand therefore have a lower melt temperature than the primers.Accordingly, the melt temperature of the amplicon produced by theprimers should also have a higher melt temperature than the probes.Thus, as the mixture cools, the re-formation of the double strandedamplicon would be expected. As previously stated, however, this is notthe case. The probes are found to preferentially bind the singlestranded amplicon members. Moreover, this preference of probe/singlestranded amplicon binding exists even when the primer sequences areadded in excess of the probes.

After the probe/single stranded amplicon member hybrids are formed, theyare detected. Standard heterogeneous assay formats are suitable fordetecting the hybrids using the detection labels and capture labelspresent on the primers and probes. The hybrids can be bound to a solidphase reagent by virtue of the capture label and detected by virtue ofthe detection label. In cases where the detection label is directlydetectable, the presence of the hybrids on the solid phase can bedetected by causing the label to produce a detectable signal, ifnecessary, and detecting the signal. In cases where the label is notdirectly detectable, the captured hybrids can be contacted with aconjugate, which generally comprises a binding member attached to adirectly detectable label. The conjugate becomes bound to the complexesand the conjugates presence on the complexes can be detected with thedirectly detectable label. Thus, the presence of the hybrids on thesolid phase reagent can be determined. Those skilled in the art willrecognize that wash steps may be employed to wash away unhybridizedamplicon or probe as well as unbound conjugate.

Although the target sequence is described as single stranded, it also iscontemplated to include the case where the target sequence is actuallydouble stranded but is merely separated from its complement prior tohybridization with the amplification primer sequences. In the case wherePCR is employed in this method, the ends of the target sequences areusually known. In cases where LCR or a modification thereof is employedin the preferred method, the entire target sequence is usually known.Typically, the target sequence is a nucleic acid sequence such as, forexample, RNA or DNA.

The method provided herein can be used in well-known amplificationreactions that include thermal cycle reaction mixtures, particularly inPCR and gap LCR (GLCR). Amplification reactions typically employ primersto repeatedly generate copies of a target nucleic acid sequence, whichtarget sequence is usually a small region of a much larger nucleic acidsequence. Primers are themselves nucleic acid sequences that arecomplementary to regions of a target sequence. Under amplificationconditions, these primers hybridize or bind to the complementary regionsof the target sequence. Copies of the target sequence typically aregenerated by the process of primer extension and/or ligation whichutilizes enzymes with polymerase or ligase activity, separately or incombination, to add nucleotides to the hybridized primers and/or ligateadjacent probe pairs. The nucleotides that are added to the primers orprobes, as monomers or preformed oligomers, are also complementary tothe target sequence. Once the primers or probes have been sufficientlyextended and/or ligated, they are separated from the target sequence,for example, by heating the reaction mixture to a “melt temperature”which is one in which complementary nucleic acid strands dissociate.Thus, a sequence complementary to the target sequence is formed.

A new amplification cycle then can take place to further amplify thenumber of target sequences by separating any double stranded sequences,allowing primers or probes to hybridize to their respective targets,extending and/or ligating the hybridized primers or probes andre-separating. The complementary sequences that are generated byamplification cycles can serve as templates for primer extension orfilling the gap of two probes to further amplify the number of targetsequences. Typically, a reaction mixture is cycled between 20 and 100times, more typically, a reaction mixture is cycled between 25 and 50times. The numbers of cycles can be determined by the routineer. In thismanner, multiple copies of the target sequence and its complementarysequence are produced. Thus, primers initiate amplification of thetarget sequence when it is present under amplification conditions.

Generally, two primers which are complementary to a portion of a targetstrand and its complement are employed in PCR. For LCR, four probes, twoof which are complementary to a target sequence and two of which aresimilarly complementary to the target's complement, are generallyemployed. In addition to the primer sets and enzymes previouslymentioned, a nucleic acid amplification reaction mixture may alsocomprise other reagents which are well known and include but are notlimited to: enzyme cofactors such as manganese; magnesium; salts;nicotinamide adenine dinucleotide (NAD); and deoxynucleotidetriphosphates (dNTPs) such, as for example, deoxyadenine triphosphate,deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythyminetriphosphate.

While the amplification primers initiate amplification of the targetsequence, the detection (or hybridization) probe is not involved inamplification. Detection probes are generally nucleic acid sequences oruncharged nucleic acid analogs such as, for example, peptide nucleicacids which are disclosed in International Publication No. WO 92/20702;morpholino analogs which are described in U.S. Pat. Nos. 5,185,444,5,034,506 and 5,142,047; and the like. Depending upon the type of labelcarried by the probe, the probe is employed to capture or detect theamplicon generated by the amplification reaction. The probe is notinvolved in amplification of the target sequence and therefore may haveto be rendered “non-extendible” in that additional dNTPs cannot be addedto the probe. In and of themselves analogs usually are non-extendibleand nucleic acid probes can be rendered non-extendible by modifying the3′ end of the probe such that the hydroxyl group is no longer capable ofparticipating in elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified. U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 and incorporated herein byreference describes modifications which can be used to render a probenon-extendible.

The ratio of primers to probes is not important. Thus, either the probesor primers can be added to the reaction mixture in excess whereby theconcentration of one would be greater than the concentration of theother. Alternatively, primers and probes can be employed in equivalentconcentrations. Preferably, however, the primers are added to thereaction mixture in excess of the probes. Thus, primer to probe ratiosof, for example, 5:1 and 20:1 are preferred.

While the length of the primers and probes can vary, the probe sequencesare selected such that they have a lower melt temperature than theprimer sequences. Hence, the primer sequences are generally longer thanthe probe sequences. Typically, the primer sequences are in the range ofbetween 20 and 50 nucleotides long, more typically in the range ofbetween 20 and 30 nucleotides long. The typical probe is in the range ofbetween 10 and 25 nucleotides long.

Various methods for synthesizing primers and probes are well known inthe art. Similarly, methods for attaching labels to primers or probesare also well known in the art. For example, it is a matter of routineto synthesize desired nucleic acid primers or probes using conventionalnucleotide phosphoramidite chemistry and instruments available fromApplied Biosystems, Inc., (Foster City, Calif.), Dupont (Wilmington,Del.), or Milligen (Bedford Mass.). Many methods have been described forlabeling oligonucleotides such as the primers or probes of the presentinvention. Enzo Biochemical (New York, N.Y.) and Clontech (Palo Alto,Calif.) both have described and commercialized probe labelingtechniques. For example, a primary amine can be attached to a 3′ oligoterminus using 3′-Amine-ON CPG™ (Clontech, Palo Alto, Calif.).Similarly, a primary amine can be attached to a 5′ oligo terminus usingAminomodifier II® (Clontech). The amines can be reacted to varioushaptens using conventional activation and linking chemistries. Inaddition, copending applications U.S. Ser. Nos. 625,566, filed Dec. 11,1990 and 630,908, filed Dec. 20, 1990, which are each incorporatedherein by reference, teach methods for labeling probes at their 5′ and3′ termini, respectively. International Publication Nos WO 92/10505,published Jun. 25, 1992, and WO 92/11388, published Jul. 9, 1992, teachmethods for labeling probes at their 5′ and 3′ ends, respectively.According to one known method for labeling an oligonucleotide, alabel-phosphoramidite reagent is prepared and used to add the label tothe oligonucleotide during its synthesis. See, for example, N. T. Thuonget al., Tet. Letters 29(46):5905-5908 (1988); or J. S. Cohen et al.,published U.S. patent application Ser. No. 07/246,688 (NTIS ORDER No.PAT-APPL-7-246,688) (1989). Preferably, probes are labeled at their 3′and 5′ ends.

A capture label is attached to the primers or probes and can be aspecific binding member which forms a binding pair with the solid phasereagent's specific binding member. It will be understood that the primeror probe itself may serve as the capture label. For example, in the casewhere a solid phase reagent's binding member is a nucleic acid sequence,it may be selected such that it binds a complementary portion of theprimer or probe to thereby immobilize the primer or probe to the solidphase. In cases where the probe itself serves as the binding member,those skilled in the art will recognize that the probe will contain asequence or “tail” that is not complementary to the single strandedamplicon members. In the case where the primer itself serves as thecapture label, at least a portion of the primer will be free tohybridize with a nucleic acid on a solid phase because the probe isselected such that it is not fully complementary to the primer sequence.

Generally, probe/single stranded amplicon member complexes can bedetected using techniques commonly employed to perform heterogeneousimmunoassays. Preferably, in this embodiment, detection is performedaccording to the protocols used by the commercially available AbbottLCx® instrumentation (Abbott Laboratories, Abbott Park, Ill.).

The primers and probes disclosed herein are useful in typical PCRassays, wherein the test sample is contacted with a pair of primers,amplification is performed, the hybridization probe is added, anddetection is performed.

Another method provided by the present invention comprises contacting atest sample with a plurality of polynucleotides, wherein at least onepolynucleotide is a BU101 molecule as described herein, hybridizing thetest sample with the plurality of polynucleotides and detectinghybridization complexes. Hybridization complexes are identified andquantitated to compile a profile which is indicative of breast tissuedisease, such as breast cancer. Expressed RNA sequences may further bedetected by reverse transcription and amplification of the DNA productby procedures well-known in the art, including polymerase chain reaction(PCR).

Drug Screening and Gene Therapy

The present invention also encompasses the use of gene therapy methodsfor the introduction of anti-sense BU101 derived molecules, such aspolynucleotides or oligonucleotides of the present invention, intopatients with conditions associated with abnormal expression ofpolynucleotides related to a breast tissue disease or conditionespecially breast cancer. These molecules, including antisense RNA andDNA fragments and ribozymes, are designed to inhibit the translation ofBU101-mRNA, and may be used therapeutically in the treatment ofconditions associated with altered or abnormnal expression of a BU101polynucleotide.

Alternatively, the oligonucleotides described above can be delivered tocells by procedures known in the art such that the anti-sense RNA or DNAmay be expressed in vivo to inhibit production of a BU101 polypeptide inthe manner described above. Antisense constructs to a BU101polynucleotide, therefore, reverse the action of BU101 transcripts andmay be used for treating breast tissue disease conditions, such asbreast cancer. These antisense constructs may also be used to treattumor metastases.

The present invention also provides a method of screening a plurality ofcompounds for specific binding to BU101 polypeptide(s), or any fragmentthereof, to identify at least one compound which specifically binds theBU101 polypeptide. Such a method comprises the steps of providing atleast one compound; combining the BU101 polypeptide with each compoundunder suitable conditions for a time sufficient to allow binding; anddetecting the BU101 polypeptide binding to each compound.

The polypeptide or peptide fragment employed in such a test may eitherbe free in solution, affixed to a solid support, borne on a cell surfaceor located intracellularly. One method of drug screening utilizeseukaryotic or prokaryotic host cells which are stably transfected withrecombinant nucleic acids which can express the polypeptide or peptidefragment. Drugs may be screened against such transfected cells incompetitive binding assays. For example, the formation of complexesbetween a polypeptide and the agent being tested can be measured ineither viable or fixed cells.

The present invention thus provides methods of screening for drugs orany other agent which can be used to treat diseases associated withBU101. These methods comprise contacting the drug with a polypeptide orfragment thereof and assaying for either the presence of a complexbetween the agent and the polypeptide, or for the presence of a complexbetween the polypeptide and the cell. In competitive binding assays, thepolypeptide typically is labeled. After suitable incubation, free (oruncomplexed) polypeptide or fragment thereof is separated from thatpresent in bound form, and the amount of free or uncomplexed label isused as a measure of the ability of the particular drug to bind to thepolypeptide or to interfere with the polypeptide/cell complex.

The present invention also encompasses the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptide specifically compete with a test drug for binding to thepolypeptide or fragment thereof. In this manner, the antibodies can beused to detect the presence of any polypeptide in the test sample whichshares one or more antigenic determinants with a BU101 polypeptide asprovided herein.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to at least onepolypeptide of BU101 disclosed herein. Briefly, large numbers ofdifferent small peptide test compounds are synthesized on a solid phase,such as plastic pins or some other surface. The peptide test compoundsare reacted with polypeptide and washed. Polypeptide thus bound to thesolid phase is detected by methods well-known in the art. Purifiedpolypeptide can also be coated directly onto plates for use in the drugscreening techniques described herein. In addition, non-neutralizingantibodies can be used to capture the polypeptide and immobilize it onthe solid support. See, for example, EP 84/03564, published on Sep. 13,1984, which is incorporated herein by reference.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of the small moleculesincluding agonists, antagonists, or inhibitors with which they interact.Such structural analogs can be used to design drugs which are moreactive or stable forms of the polypeptide or which enhance or interferewith the function of a polypeptide in vivo. J. Hodgson, Bio/Technology9:19-21 (1991), incorporated herein by reference.

For example, in one approach, the three-dimensional structure of apolypeptide, or of a polypeptide-inhibitor complex, is determined byx-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous polypeptide-likemolecules or to identify efficient inhibitors

Useful examples of rational drug design may include molecules which haveimproved activity or stability as shown by S. Braxton et al.,Biochemistry 31:7796-7801 (1992), or which act as inhibitors, agonists,or antagonists of native peptides as shown by S. B. P. Athauda et al.,J. Biochem. (Tokyo) 113 (6):742-746 (1993), incorporated herein byreference.

It also is possible to isolate a target-specific antibody selected by anassay as described hereinabove, and then to determine its crystalstructure. In principle, this approach yields a pharmacophore upon whichsubsequent drug design can be based. It further is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (“anti-ids”) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-id is an analog of the original receptor. The anti-id then can beused to identify and isolate peptides from banks of chemically orbiologically produced peptides. The isolated peptides then can act asthe pharmacophore (that is, a prototype pharmaceutical drug).

A sufficient amount of a recombinant polypeptide of the presentinvention may be made available to perform analytical studies such asX-ray crystallography. In addition, knowledge of the polypeptide aminoacid sequence which is derivable from the nucleic acid sequence providedherein will provide guidance to those employing computer modelingtechniques in place of, or in addition to, x-ray crystallography.

Antibodies specific to a BU101 polypeptide (e.g., anti-BU101 antibodies)further may be used to inhibit the biological action of the polypeptideby binding to the polypeptide. In this manner, the antibodies may beused in therapy, for example, to treat breast tissue diseases includingbreast cancer and its metastases.

Further, such antibodies can detect the presence or absence of the BU101polypeptide in a test sample and, therefore, are useful as diagnosticmarkers for the diagnosis of a breast tissue disease or conditionespecially breast cancer. Such antibodies may also function as adiagnostic marker for breast tissue disease conditions such as breastcancer. The present invention also is directed to antagonists andinhibitors of the polypeptides of the present invention. The antagonistsand inhibitors are those which inhibit or eliminate the function of thepolypeptide. Thus, for example, an antagonist may bind to a polypeptideof the present invention and inhibit or eliminate its function. Theantagonist, for example, could be an antibody against the polypeptidewhich eliminates the activity of the BU101 polypeptide by binding theBU101 polypeptide, or in some cases the antagonist may be anoligonucleotide. Examples of small molecule inhibitors include, but arenot limited to, small peptides or peptide-like molecules.

The antagonists and inhibitors may be employed as a composition with apharmaceutically acceptable carrier, including, but not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. Administration of BU101 polypeptide inhibitors ispreferably systemic. The present invention also provides an antibodywhich inhibits the action of such a polypeptide.

Antisense technology can be used to reduce gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes thepolypeptide of the present invention, is used to design an antisense RNAoligonucleotide of from 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription, thereby preventing transcription and theproduction of the BU101 polypeptide. For triple helix, see, for example,Lee et al, Nuc. Acids Res. 6:3073 (1979); Cooney et al, Science 241:456(1988); and Dervan et al, Science 251:1360 (1991) The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofa mRNA molecule into the BU101 polypeptide. For antisense, see, forexample, Okano, J. Neurochem. 56:560 (1991); and “Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression”, CRC Press, Boca Raton, Fla.(1988). Antisense oligonucleotides act with greater efficacy whenmodified to contain artificial internucleotide linkages which render themolecule resistant to nucleolytic cleavage. Such artificialinternucleotide linkages include, but are not limited to,methylphosphonate, phosphorothiolate and phosphoroamydateinternucleotide linkages.

Recombinant Technology

The present invention provides host cells and expression vectorscomprising BU101 polynucleotides of the present invention and methodsfor the production of the polypeptide(s) they encode. Such methodscomprise culturing the host cells under conditions suitable for theexpression of the BU101 polynucleotide and recovering the BU101polypeptide from the cell culture.

The present invention also provides vectors which include BU101polynucleotides of the present invention, host cells which aregenetically engineered with vectors of the present invention and theproduction of polypeptides of the present invention by recombinanttechniques.

Host cells are genetically engineered (transfected, transduced ortransformed) with the vectors of this invention which may be cloningvectors or expression vectors. The vector may be in the form of aplasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transfected cells, or amplifying BU101gene(s). The culture conditions, such as temperature, pH and the like,are those previously used with the host cell selected for expression,and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, thepolynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virusand pseudorabies. However, any other plasmid or vector may be used solong as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted intoappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art. The DNA sequence in the expression vector isoperatively linked to an appropriate expression control sequence(s)(promoter) to direct MRNA synthesis. Representative examples of suchpromoters include, but are not limited to, the LTR or the SV40 promoter,the E. coli lac or trp, the phage lambda P sub L promoter and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vector also contains aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In addition, the expression vectors preferablycontain a gene to provide a phenotypic trait for selection oftransfected host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transfect an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium; Streptomyces sp; fungal cells, such as yeast; insect cellssuch as Drosophila and Sf9; animal cells such as CHO, COS or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings provided herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. The following vectorsare provided by way of example. Bacterial: pINCY (Incyte PharmaceuticalsInc., Palo Alto, Calif.), pSPORT1 (Life Technologies, Gaithersburg,Md.), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia). However, any other plasmid or vector may be used as long asit is replicable and viable in the host.

Plasmid pINCY is generally identical to the plasmid pSPORT1 (availablefrom Life Technologies, Gaithersburg, Md.) with the exception that ithas two modifications in the polylinker (multiple cloning site). Thesemodifications are (1) it lacks a HindIII restriction site and (2) itsEcoRI restriction site lies at a different location. pINCY is createdfrom pSPORT1 by cleaving pSPORT1 with both HindIII and EcoRI andreplacing the excised fragment of the polylinker with synthetic DNAfragments (SEQUENCE ID NO 5 and SEQUENCE ID NO 6). This replacement maybe made in any manner known to those of ordinary skill in the art. Forexample, the two nucleotide sequences, SEQUENCE ID NO 5 and SEQUENCE IDNO 6, may be generated synthetically with 5′ terminal phosphates, mixedtogether, and then ligated under standard conditions for performingstaggered end ligations into the pSPORT1 plasmid cut with HindIII andEcoRI. Suitable host cells (such as E. coli DH5∝cells) then aretransfected with the ligated DNA and recombinant clones are selected forampicillin resistance. Plasmid DNA then is prepared from individualclones and subjected to restriction enzyme analysis or DNA sequencing inorder to confirm the presence of insert sequences in the properorientation. Other cloning strategies known to the ordinary artisan alsomay be employed.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, SP6, T7, gpt, lambda P subR, P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, LTRs from retroviruses and mouse metallothionein-I. Selectionof the appropriate vector and promoter is well within the level ofordinary skill in the art.

In a further embodiment, the present invention provides host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (L. Davis et al., “Basic Methods inMolecular Biology”, 2nd edition, Appleton and Lang, ParamountPublishing, East Norwalk, Conn. (1994)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Recombinant proteins can be expressed in mammalian cells, yeast,bacteria, or other cells, under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptide(s) of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication originand adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transfection of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransfection include E. coli, Bacillus subtilis, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, although, others may also be employed as a routinematter of choice.

Useful expression vectors for bacterial use comprise a selectable markerand bacterial origin of replication derived from plasmids comprisinggenetic elements of the well-known cloning vector pBR322 (ATCC 37017).Other vectors include but are not limited to PKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.).These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transfection of a suitable host and growth of the host to anappropriate cell density, the selected promoter is derepressed byappropriate means (e.g., temperature shift or chemical induction), andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents; such methods arewell-known to the ordinary artisan.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, such as the C 127, HEK-293, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements. Representative, useful vectors include pRc/CMV andpcDNA3 (available from Invitrogen, San Diego, Calif.).

BU101 polypeptides are recovered and purified from recombinant cellcultures by known methods including affinity chromatography, ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, hydroxyapatite chromatography or lectinchromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price, et al., J. Biol. Chem. 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of thepolypeptide. Finally, high performance liquid chromatography (HPLC) canbe employed for final purification steps.

Thus, polypeptides of the present invention may be naturally purifiedproducts expressed from a high expressing cell line, or a product ofchemical synthetic procedures, or produced by recombinant techniquesfrom a prokaryotic or eukaryotic host (for example, by bacterial, yeast,higher plant, insect and mammalian cells in culture). Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated with mammalian or othereukaryotic carbohydrates or may be non-glycosylated. The polypeptides ofthe invention may also include an initial methionine amino acid residue.

The starting plasmids can be constructed from available plasmids inaccord with published, known procedures. In addition, equivalentplasmids to those described are known in the art and will be apparent tothe ordinarily skilled artisan.

The following is the general procedure for the isolation and analysis ofcDNA clones. In a particular embodiment disclosed herein, mRNA wasisolated from breast tissue and used to generate the cDNA library.Breast tissue was obtained from patients by surgical resection and wasclassified as tumor or non-tumor tissue by a pathologist.

The cDNA inserts from random isolates of the breast tissue librarieswere sequenced in part, analyzed in detail as set forth in the Examplesand are disclosed in the Sequence Listing as SEQUENCE ID NO 1 andSEQUENCE ID NO 2. The consensus sequence of these inserts is presentedas SEQUENCE ID NO 3. These polynucleotides may contain an entire openreading frame with or without associated regulatory sequences for aparticular gene, or they may encode only a portion of the gene ofinterest. This is attributed to the fact that many genes are severalhundred and sometimes several thousand, bases in length and, withcurrent technology, cannot be cloned in their entirety because of vectorlimitations, incomplete reverse transcription of the first strand, orincomplete replication of the second strand. Contiguous, secondaryclones containing additional nucleotide sequences may be obtained usinga variety of methods known to those of skill in the art.

Methods for DNA sequencing are well known in the art. Conventionalenzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (USBiochemical Corp, Cleveland, Ohio) or Taq polymerase to extend DNAchains from an oligonucleotide primer annealed to the DNA template ofinterest. Methods have been developed for the use of bothsingle-stranded and double-stranded templates. The chain terminationreaction products may be electrophoresed on urea/polyacrylamide gels anddetected either by autoradiography (for radionucleotide labeledprecursors) or by fluorescence (for fluorescent-labeled precursors).Recent improvements in mechanized reaction preparation, sequencing andanalysis using the fluorescent detection method have permitted expansionin the number of sequences that can be determined per day using machinessuch as the Applied Biosystems 377 DNA Sequencers (Applied Biosystems,Foster City, Calif.).

The reading frame of the nucleotide sequence can be ascertained byseveral types of analyses. First, reading frames contained within thecoding sequence can be analyzed for the presence of start codon ATG andstop codons TGA, TAA or TAG. Typically, one reading frame will continuethroughout the major portion of a cDNA sequence while other readingframes tend to contain numerous stop codons. In such cases, readingframe determination is straightforward. In other more difficult cases,further analysis is required.

Algorithms have been created to analyze the occurrence of individualnucleotide bases at each putative codon triplet. See, for example J. W.Fickett, Nuc Acids Res 10:5303 (1982). Coding DNA for particularorganisms (bacteria, plants and animals) tends to contain certainnucleotides within certain triplet periodicities, such as a significantpreference for pyrimidines in the third codon position. Thesepreferences have been incorporated into widely available software whichcan be used to determine coding potential (and frame) of a given stretchof DNA. The algorithm-derived information combined with start/stop codoninformation can be used to determine proper frame with a high degree ofcertainty. This, in turn, readily permits cloning of the sequence in thecorrect reading frame into appropriate expression vectors.

The nucleic acid sequences disclosed herein may be joined to a varietyof other polynucleotide sequences and vectors of interest by means ofwell-established recombinant DNA techniques. See J. Sambrook et al.,supra. Vectors of interest include cloning vectors, such as plasmids,cosmids, phage derivatives, phagemids, as well as sequencing,replication and expression vectors, and the like. In general, suchvectors contain an origin of replication functional in at least oneorganism, convenient restriction endonuclease digestion sites andselectable markers appropriate for particular host cells. The vectorscan be transferred by a variety of means known to those of skill in theart into suitable host cells which then produce the desired DNA, RNA orpolypeptides.

Occasionally, sequencing or random reverse transcription errors willmask the presence of the appropriate open reading frame or regulatoryelement. In such cases, it is possible to determine the correct readingframe by attempting to express the polypeptide and determining the aminoacid sequence by standard peptide mapping and sequencing techniques.See, F. M. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y. (1989). Additionally, the actual readingframe of a given nucleotide sequence may be determined by transfectionof host cells with vectors containing all three potential readingframes. Only those cells with the nucleotide sequence in the correctreading frame will produce a peptide of the predicted length.

The nucleotide sequences provided herein have been prepared by current,state-of-the-art, automated methods and as such may contain unidentifiednucleotides. These will not present a problem to those skilled in theart who wish to practice the invention. Several methods employingstandard recombinant techniques, described in J. Sambrook (supra) orperiodic updates thereof, may be used to complete the missing sequenceinformation. The same techniques used for obtaining a full lengthsequence, as described herein, may be used to obtain nucleotidesequences.

Expression of a particular cDNA may be accomplished by subcloning thecDNA into an appropriate expression vector and transfecting this vectorinto an appropriate expression host. The cloning vector used for thegeneration of the breast tissue cDNA library can be used fortranscribing mRNA of a particular cDNA and contains a promoter forbeta-galactosidase, an amino-terminal met and the subsequent seven aminoacid residues of beta-galactosidase. Immediately following these eightresidues is an engineered bacteriophage promoter, useful for artificialpriming and transcription, as well as a number of unique restrictionsites, including EcoRI, for cloning. The vector can be transfected intoan appropriate host strain of E. coli.

Induction of the isolated bacterial strain with isopropylthiogalactoside(IPTG) using standard methods will produce a fusion protein whichcontains the first seven residues of beta-galactosidase, about 15residues of linker and the peptide encoded within the cDNA. Since cDNAclone inserts are generated by an essentially random process, there isone chance in three that the included cDNA will lie in the correct framefor proper translation. If the cDNA is not in the proper reading frame,the correct frame can be obtained by deletion or insertion of anappropriate number of bases by well known methods including in vitromutagenesis, digestion with exonuclease III or mung bean nuclease, oroligonucleotide linker inclusion.

The cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide primers,containing cloning sites and segments of DNA sufficient to hybridize tostretches at both ends of the target cDNA, can be synthesized chemicallyby standard methods. These primers can then be used to amplify thedesired gene segments by PCR. The resulting new gene segments can bedigested with appropriate restriction enzymes under standard conditionsand isolated by gel electrophoresis. Alternately, similar gene segmentscan be produced by digestion of the cDNA with appropriate restrictionenzymes and filling in the missing gene segments with chemicallysynthesized oligonucleotides. Segments of the coding sequence from morethan one gene can be ligated together and cloned in appropriate vectorsto optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to, mammalian cells such as Chinese Hamster Ovary (CHO) andhuman embryonic kidney (HEK) 293 cells, insect cells such as Sf9 cells,yeast cells such as Saccharomyces cerevisiae and bacteria such as E.coli. For each of these cell systems, a useful expression vector mayalso include an origin of replication to allow propagation in bacteriaand a selectable marker such as the beta-lactamase antibiotic resistancegene to allow selection in bacteria. In addition, the vectors mayinclude a second selectable marker, such as the neomycinphosphotransferase gene, to allow selection in transfected eukaryotichost cells. Vectors for use in eukaryotic expression hosts may requirethe addition of 3′ poly A tail if the sequence of interest lacks poly A.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and include,but are not limited to, MMTV, SV40, or metallothionine promoters for CHOcells; trp, lac, tac or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase or PGH promoters for yeast. Adenoviral vectorswith or without transcription enhancers, such as the rous sarcoma virus(RSV) enhancer, may be used to drive protein expression in mammaliancell lines. Once homogeneous cultures of recombinant cells are obtained,large quantities of recombinantly produced protein can be recovered fromthe conditioned medium and analyzed using chromatographic methods wellknown in the art. An alternative method for the production of largeamounts of secreted protein involves the transfection of mammalianembryos and the recovery of the recombinant protein from milk producedby transgenic cows, goats, sheep, etc. Polypeptides and closely relatedmolecules may be expressed recombinantly in such a way as to facilitateprotein purification. One approach involves expression of a chimericprotein which includes one or more additional polypeptide domains notnaturally present on human polypeptides. Such purification-facilitatingdomains include, but are not limited to, metal-chelating peptides suchas histidine-tryptophan domains that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase fromInvitrogen (San Diego, Calif.) between the polypeptide sequence and thepurification domain may be useful for recovering the polypeptide.

Immunoassays

BU101 polypeptides, including fragments, derivatives, and analogsthereof, or cells expressing such polypeptides, can be utilized in avariety of assays, many of which are described herein, for the detectionof antibodies to breast tissue. They also can be used as immunogens toproduce antibodies. These antibodies can be, for example, polyclonal ormonoclonal antibodies, chimeric, single chain and humanized antibodies,as well as Fab fragments, or the product of an Fab expression library.Various procedures known in the art may be used for the production ofsuch antibodies and fragments.

For example, antibodies generated against a polypeptide comprising asequence of the present invention can be obtained by direct injection ofthe polypeptide into an animal or by administering the polypeptide to ananimal such as a mouse, rabbit, goat or human. A mouse, rabbit or goatis preferred. The polypeptide is selected from the group consisting ofSEQUENCE ID NOS 15-23, and fragments thereof. The antibody so obtainedthen will bind the polypeptide itself. In this manner, even a sequenceencoding only a fragment of the polypeptide can be used to generateantibodies that bind the native polypeptide. Such antibodies then can beused to isolate the polypeptide from test samples such as tissuesuspected of containing that polypeptide. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique as described by Kohler and Milstein, Nature256:495-497 (1975), the trioma technique, the human B-cell hybridomatechnique as described by Kozbor et al, Immun. Today 4:72 (1983) and theEBV-hybridoma technique to produce human monoclonal antibodies asdescribed by Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc, New York, N.Y., pp. 77-96 (1985). Techniquesdescribed for the production of single chain antibodies can be adaptedto produce single chain antibodies to immunogenic polypeptide productsof this invention. See, for example, U.S. Pat. No. 4,946,778, which isincorporated herein by reference.

Various assay formats may utilize the antibodies of the presentinvention, including “sandwich” immunoassays and probe assays. Forexample, the antibodies of the present invention, or fragments thereof,can be employed in various assay systems to determine the presence, ifany, of BU101 antigen in a test sample. For example, in a first assayformat, a polyclonal or monoclonal antibody or fragment thereof, or acombination of these antibodies, which has been coated on a solid phase,is contacted with a test sample, to form a first mixture. This firstmixture is incubated for a time and under conditions sufficient to formantigen/antibody complexes. Then, an indicator reagent comprising amonoclonal or a polyclonal antibody or a fragment thereof, or acombination of these antibodies, to which a signal generating compoundhas been attached, is contacted with the antigen/antibody complexes toform a second mixture. This second mixture then is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence of BU101 antigen in the test sample and capturedon the solid phase, if any, is determined by detecting the measurablesignal generated by the signal generating compound. The amount of BU101antigen present in the test sample is proportional to the signalgenerated.

In an alternative assay format, a mixture is formed by contacting: (1) apolyclonal antibody, monoclonal antibody, or fragment thereof, whichspecifically binds to BU101 antigen, or a combination of such antibodiesbound to a solid support; (2) the test sample; and (3) an indicatorreagent comprising a monoclonal antibody, polyclonal antibody, orfragment thereof, which specifically binds to a different BU101 antigen(or a combination of these antibodies) to which a signal generatingcompound is attached. This mixture is incubated for a time and underconditions sufficient to form antibody/antigen/antibody complexes. Thepresence, if any, of BU101 antigen present in the test sample andcaptured on the solid phase is determined by detecting the measurablesignal generated by the signal generating compound. The amount of BU101antigen present in the test sample is proportional to the signalgenerated.

In another assay format, one or a combination of at least two monoclonalantibodies of the invention can be employed as a competitive probe forthe detection of antibodies to BU101 antigen. For example, BU101polypeptides such as the recombinant antigens disclosed herein, eitheralone or in combination, are coated on a solid phase. A test samplesuspected of containing antibody to BU101 antigen then is incubated withan indicator reagent comprising a signal generating compound and atleast one monoclonal antibody of the invention for a time and underconditions sufficient to form antigen/antibody complexes of either thetest sample and indicator reagent bound to the solid phase or theindicator reagent bound to the solid phase. The reduction in binding ofthe monoclonal antibody to the solid phase can be quantitativelymeasured.

In yet another detection method, each of the monoclonal or polyclonalantibodies of the present invention can be employed in the detection ofBU101 antigens in tissue sections, as well as in cells, byimmunohistochemical analysis. Cytochemical analysis wherein theseantibodies are labeled directly (with, for example, fluorescein,colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) orare labeled by using secondary labeled anti-species antibodies (withvarious labels as exemplified herein) to track the histopathology ofdisease also are within the scope of the present invention.

In addition, these monoclonal antibodies can be bound to matricessimilar to CNBr-activated Sepharose and used for the affinitypurification of specific BU101 polypeptides from cell cultures orbiological tissues such as to purify recombinant and native BU101proteins.

The monoclonal antibodies of the invention also can be used for thegeneration of chimeric antibodies for therapeutic use, or other similarapplications.

The monoclonal antibodies or fragments thereof can be providedindividually to detect BU101 antigens. Combinations of the monoclonalantibodies (and fragments thereof) provided herein also may be usedtogether as components in a mixture or “cocktail” of at least one BU101antibody of the invention, along with antibodies which specifically bindto other BU101 regions, each antibody having different bindingspecificities. Thus, this cocktail can include the monoclonal antibodiesof the invention which are directed to BU101 polypeptides disclosedherein and other monoclonal antibodies specific to other antigenicdeterminants of BU101 antigens or other related proteins.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to a BU101 polypeptide or otherBU101 polypeptides additionally used in the assay. The polyclonalantibody used preferably is of mammalian origin such as, human, goat,rabbit or sheep polyclonal antibody which binds BU101 polypeptide. Mostpreferably, the polyclonal antibody is of rabbit origin. The polyclonalantibodies used in the assays can be used either alone or as a cocktailof polyclonal antibodies. Since the cocktails used in the assay formatsare comprised of either monoclonal antibodies or polyclonal antibodieshaving different binding specificity to BU101 polypeptides, they areuseful for the detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining thepredisposition to, diseases and conditions of the breast such as breastcancer.

It is contemplated and within the scope of the present invention thatBU101 antigen may be detectable in assays by use of a recombinantantigen as well as by use of a synthetic peptide or purified peptide,which peptide comprises an amino acid sequence of BU101. The amino acidsequence of such a polypeptide is selected from the group consisting ofSEQUENCE ID NOS 15-23, and fragments thereof. It also is within thescope of the present invention that different synthetic, recombinant orpurified peptides, identifying different epitopes of BU101, can be usedin combination in an assay for the detecting, diagnosing, staging,monitoring, prognosticating, preventing or treating, or determining thepredisposition to diseases and conditions of the breast such as breastcancer. In this case, all of these peptides can be coated onto one solidphase; or each separate peptide may be coated onto separate solidphases, such as microparticles, and then combined to form a mixture ofpeptides which can be later used in assays. Furthermore, it iscontemplated that multiple peptides which define epitopes from differentantigens may be used for the detection, diagnosis, staging, monitoring,prognosis, prevention or treatment of, or determining the predispositionto, diseases and conditions of the breast, such as breast cancer.Peptides coated on solid phases or labeled with detectable labels arethen allowed to compete with those present in a patient sample (if any)for a limited amount of antibody. A reduction in binding of thesynthetic, recombinant, or purified peptides to the antibody (orantibodies) is an indication of the presence of BU101 antigen in thepatient sample. The presence of BU101 antigen indicates the presence ofbreast tissue disease, especially breast cancer, in the patient.Variations of assay formats are known to those of ordinary skill in theart and many are discussed herein below.

In another assay format, the presence of anti-BU101 antibody and/orBU101 antigen can be detected in a simultaneous assay, as follows. Atest sample is simultaneously contacted with a capture reagent of afirst analyte, wherein said capture reagent comprises a first bindingmember specific for a first analyte attached to a solid phase and acapture reagent for a second analyte, wherein said capture reagentcomprises a first binding member for a second analyte attached to asecond solid phase, to thereby form a mixture. This mixture is incubatedfor a time and under conditions sufficient to form capture reagent/firstanalyte and capture reagent/second analyte complexes. These so-formedcomplexes then are contacted with an indicator reagent comprising amember of a binding pair specific for the first analyte labeled with asignal generating compound and an indicator reagent comprising a memberof a binding pair specific for the second analyte labeled with a signalgenerating compound to form a second mixture. This second mixture isincubated for a time and under conditions sufficient to form capturereagent/first analyte/indicator reagent complexes and capturereagent/second analyte/indicator reagent complexes. The presence of oneor more analytes is determined by detecting a signal generated inconnection with the complexes formed on either or both solid phases asan indication of the presence of one or more analytes in the testsample. In this assay format, recombinant antigens derived from theexpression systems disclosed herein may be utilized, as well asmonoclonal antibodies produced from the proteins derived from theexpression systems as disclosed herein. For example, in this assaysystem, BU101 antigen can be the first analyte. Such assay systems aredescribed in greater detail in EP Publication No. 0473065.

In yet other assay formats, the polypeptides disclosed herein may beutilized to detect the presence of antibody against BU101 antigen intest samples. For example, a test sample is incubated with a solid phaseto which at least one polypeptide such as a recombinant protein orsynthetic peptide has been attached. The polypeptide is selected fromthe group consisting of SEQUENCE ID NOS 15-23, and fragments thereof.These are reacted for a time and under conditions sufficient to formantigen/antibody complexes. Following incubation, the antigen/antibodycomplex is detected. Indicator reagents may be used to facilitatedetection, depending upon the assay system chosen. In another assayformat, a test sample is contacted with a solid phase to which arecombinant protein produced as described herein is attached, and alsois contacted with a monoclonal or polyclonal antibody specific for theprotein, which preferably has been labeled with an indicator reagent.After incubation for a time and under conditions sufficient forantibody/antigen complexes to form, the solid phase is separated fromthe free phase, and the label is detected in either the solid or freephase as an indication of the presence of antibody against BU101antigen. Other assay formats utilizing the recombinant antigensdisclosed herein are contemplated. These include contacting a testsample with a solid phase to which at least one antigen from a firstsource has been attached, incubating the solid phase and test sample fora time and under conditions sufficient to form antigen/antibodycomplexes, and then contacting the solid phase with a labeled antigen,which antigen is derived from a second source different from the firstsource. For example, a recombinant protein derived from a first sourcesuch as E. coli is used as a capture antigen on a solid phase, a testsample is added to the so-prepared solid phase, and following standardincubation and washing steps as deemed or required, a recombinantprotein derived from a different source (i.e., non-E. coli) is utilizedas a part of an indicator reagent which subsequently is detected.Likewise, combinations of a recombinant antigen on a solid phase andsynthetic peptide in the indicator phase also are possible. Any assayformat which utilizes an antigen specific for BU101 produced or derivedfrom a first source as the capture antigen and an antigen specific forBU101 from a different second source is contemplated. Thus, variouscombinations of recombinant antigens, as well as the use of syntheticpeptides, purified proteins and the like, are within the scope of thisinvention. Assays such as this and others are described in U.S. Pat. No.5,254,458, which enjoys common ownership and is incorporated herein byreference.

Other embodiments which utilize various other solid phases also arecontemplated and are within the scope of this invention. For example,ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer (described in EP publication0326100 and EP publication No. 0406473), can be employed according tothe present invention to effect a fast solution-phase immunochemicalreaction. An immobilizable immune complex is separated from the rest ofthe reaction mixture by ionic interactions between the negativelycharged poly-anion/immune complex and the previously treated, positivelycharged porous matrix and detected by using various signal generatingsystems previously described, including those described inchemiluminescent signal measurements as described in EPO Publication No.0 273,115.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises a microparticle(magnetic or non-magnetic). Such systems include those described in, forexample, published EPO applications Nos. EP 0 425 633 and EP 0 424 634,respectively.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies of the present inventionare easily adaptable. In scanning probe microscopy, particularly inatomic force microscopy, the capture phase, for example, at least one ofthe monoclonal antibodies of the invention, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunneling microscopy eliminates the need for labelswhich normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. The use of SPM to monitor specific bindingreactions can occur in many ways. In one embodiment, one member of aspecific binding partner (analyte specific substance which is themonoclonal antibody of the invention) is attached to a surface suitablefor scanning. The attachment of the analyte specific substance may be byadsorption to a test piece which comprises a solid phase of a plastic ormetal surface, following methods known to those of ordinary skill in theart. Or, covalent attachment of a specific binding partner (analytespecific substance) to a test piece which test piece comprises a solidphase of derivatized plastic, metal, silicon, or glass may be utilized.Covalent attachment methods are known to those skilled in the art andinclude a variety of means to irreversibly link specific bindingpartners to the test piece. If the test piece is silicon or glass, thesurface must be activated prior to attaching the specific bindingpartner. Also, polyelectrolyte interactions may be used to immobilize aspecific binding partner on a surface of a test piece by usingtechniques and chemistries. The preferred method of attachment is bycovalent means. Following attachment of a specific binding member, thesurface may be further treated with materials such as serum, proteins,or other blocking agents to minimize non-specific binding. The surfacealso may be scanned either at the site of manufacture or point of use toverify its suitability for assay purposes. The scanning process is notanticipated to alter the specific binding properties of the test piece.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the reagents such as antibodies,proteins and peptides of the present invention can be utilized innon-solid phase assay systems. These assay systems are known to thoseskilled in the art, and are considered to be within the scope of thepresent invention.

It is contemplated that the reagent employed for the assay can beprovided in the form of a test kit with one or more containers such asvials or bottles, with each container containing a separate reagent suchas a probe, primer, monoclonal antibody or a cocktail of monoclonalantibodies, or a polypeptide (e.g. recombinantly, synthetically producedor purified) employed in the assay. The polypeptide is selected from thegroup consisting of SEQUENCE ID NOS 15-23, and fragments thereof. Othercomponents such as buffers, controls and the like, known to those ofordinary skill in art, may be included in such test kits. It also iscontemplated to provide test kits which have means for collecting testsamples comprising accessible body fluids, e.g., blood, urine, salivaand stool. Such tools useful for collection (“collection materials”)include lancets and absorbent paper or cloth for collecting andstabilizing blood; swabs for collecting and stabilizing saliva; cups forcollecting and stabilizing urine or stool samples. Collection materials,papers, cloths, swabs, cups and the like, may optionally be treated toavoid denaturation or irreversible adsorption of the sample. Thecollection materials also may be treated with or contain preservatives,stabilizers or antimicrobial agents to help maintain the integrity ofthe specimens. Test kits designed for the collection, stabilization andpreservation of test specimens obtained by surgery or needle biopsy arealso useful. It is contemplated that all kits may be configured in twocomponents which can be provided separately; one component forcollection and transport of the specimen and the other component for theanalysis of the specimen. The collection component, for example, can beprovided to the open market user while the components for analysis canbe provided to others such as laboratory personnel for determination ofthe presence, absence or amount of analyte. Further, kits for thecollection, stabilization and preservation of test specimens may beconfigured for use by untrained personnel and may be available in theopen market for use at home with subsequent transportation to alaboratory for analysis of the test sample.

E. coli bacteria (clone 603148) has been deposited at the American TypeCulture Collection (A.T.C.C.), 12301 Parklawn Drive, Rockville, Md.20852, as of Oct. 7, 1996, under the terms of the Budapest Treaty andwill be maintained for a period of thirty (30) years from the date ofdeposit, or for five (5) years after the last request for the deposit,or for the enforceable period of the U.S. patent, whichever is longer.The deposit and any other deposited material described herein areprovided for convenience only, and are not required to practice thepresent invention in view of the teachings provided herein. The cDNAsequence in all of the deposited material is incorporated herein byreference. Clone 603148 was accorded A.T.C.C. Deposit No. 98185.

The present invention will now be described by way of examples, whichare meant to illustrate, but not to limit, the scope of the presentinvention.

EXAMPLES Example 1 Identification of Breast Tissue Library BU101Gene-Specific Clones

A. Library Comparison of Expressed Sequence Tags (ESTs) or TranscriptImages. Partial sequences of cDNA clone inserts, so-called “expressedsequence tags” (ESTs), were derived from cDNA libraries made from breasttumor tissues, breast non-tumor tissues and numerous other tissues, bothtumor and non-tumor and entered into a database (LIFESEQ™ database,available from Incyte Pharmaceuticals, Palo Alto, Calif.) as genetranscript images. See International Publication No. WO 95/20681. (Atranscript image is a listing of the number of ESTs for each of therepresented genes in a given tissue library. ESTs sharing regions ofmutual sequence overlap are classified into clusters. A cluster isassigned a clone number from a representative 5′ EST. Often, a clusterof interest can be extended by comparing its consensus sequence withsequences of other ESTs which did not meet the criteria for automatedclustering. The alignment of all available clusters and single ESTsrepresent a contig from which a consensus sequence is derived.) Thetranscript images then were evaluated to identify EST clusters that wererepresentative primarily of the breast tissue libraries. These targetclusters then were ranked according to their abundance (occurrence ofEST members) in the target libraries and their absence from backgroundlibraries. Higher abundance clusters with low background occurrence weregiven higher study priority. A cluster, comprising 15 ESTs, wasidentified which was over-expressed in a breast tumor library, but waspresent at low levels in 3 non-tumor breast tissue libraries. No ESTswere found in any of the non-breast tissue libraries. SEQUENCE ID NO 1and SEQUENCE ID NO 2, corresponding to overlapping clones 603148 and604290, respectively, were identified for further study. Theserepresented the minimum number of clones that were needed to form theBU101 contig and from which the consensus sequence provided herein(SEQUENCE ID NO 3) was derived.

B. Generation of a Consensus Sequence. The nucleotide sequences of ESTclones, 603148 (SEQUENCE ID NO 1) and 604290 (SEQUENCE ID NO 2), wereentered in the Sequencher™ Program (available from Gene CodesCorporation, Ann Arbor, Mich., in order to generate a nucleotidealignment (contig map) and then generate their consensus sequence(SEQUENCE ID NO 3). FIG. 1 shows the nucleotide sequence alignment ofthese clones and their resultant nucleotide consensus sequence (SEQUENCEID NO 3). FIG. 2 presents the contig map depicting the clones SEQUENCEID NO 1 and SEQUENCE ID NO 2 forming overlapping regions of the BU101gene and the resultant consensus nucleotide sequence (SEQUENCE ID NO 3)of these clones in a graphic display. Following this, a three-frametranslation was performed on the consensus sequence (SEQUENCE ID NO 3).The second forward frame was found to have an open reading frameencoding a 90 residue amino acid sequence, which is presented asSEQUENCE ID NO 15. The 90 residue amino acid sequence depicted inSEQUENCE ID NO 15 was compared with published sequences using softwareand techniques known to those skilled in the art. The polypeptidesequence of a rat prostatic steroid-binding protein (psc1.pep) was foundto be partially homologous to the BU101 polypeptide of SEQUENCE ID NO15. This rat prostatic steroid-binding protein is described by Parker etal. Nature 298:92-94 (1982).

C. Specificity of Expression of ESTs Corresponding to ConsensusSequence. The consensus sequence, generated in section B, supra, wascompared to the entire updated LIFESEQ™ database (May 1997) using theBLAST search tool. ESTs corresponding to the consensus sequence werefound in 45.0% (9 of 20) of breast libraries and 0.8% (3 of 375) ofnon-breast libraries. Therefore, the consensus sequence or fragmentthereof was found more than 56 times more often in breast thannon-breast tissues.

Example 2 Sequencing of BU101 EST-Specific Clones

The DNA sequence of clone 603148 which comprises the 5′-most EST of theBU101 gene contig was determined using dideoxy termination sequencingwith dye terminators following known methods (SEQUENCE ID NO 4). (F.Sanger et al., PNAS U.S.A. 74:5463 (1977)).

Because the pSPORT1 vector (Life Technologies, Gaithersburg, Md.)contains universal priming sites just adjacent to the 3′ and 5′ ligationjunctions of the inserts, approximately 300 bases of the insert weresequenced in both directions using universal primers, SEQUENCE ID NO 7and SEQUENCE ID NO 8 (New England Biolabs, Beverly, Mass. and AppliedBiosystems Inc, Foster City, Calif., respectively). The sequencingreactions were run on a polyacrylamide denaturing gel, and the sequenceswere determined by an Applied Biosystems 377 Sequencer (available fromApplied Biosystems, Foster City, Calif.) or other sequencing apparatus.Additional sequencing primers, BU101.F1 and BU101.R1 (SEQUENCE ID NO 9and SEQUENCE ID NO 10, respectively) were designed from sequencedetermined by the initial sequencing reactions near the 3′-ends of thetwo DNA strands. These primers then were used to determine the remainingDNA sequence of the cloned insert from each DNA strand, as previouslydescribed.

Example 3 Nucleic Acid Preparation

A. RNA Extraction from Tissue. Total RNA was isolated from solid breasttissues or cells and from non-breast tissues. Various methods wereutilized, including but not limited to the lithium chloride/ureatechnique, known and described in the art (Kato et al., J. Virol.61:2182-2191, (1987)), Ultraspec™ (Biotecx Laboratories, Inc., HoustonTex.), and TRIzol™ (Life Technologies, Inc., Gaithersburg, Md.).

For northern blot analysis, the tissue was placed in a sterile conicaltube on ice and 10-15 volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 Mβ-mercaptoethanol, 50 mM Tris-HCl (pH 7.5) were added. The tissue washomogenized with a Polytron® homogenizer (Brinkman Instruments, Inc.,Westbury, N.Y.) for 30-50 sec on ice. The solution was transferred to a15 ml plastic centrifuge tube and placed overnight at −20° C. The tubewas centrifuged for 90 min at 9,000×g at 0-4° C., and the supernatantwas immediately decanted. Then, 10 ml of 3 M LiCl were added, the tubewas vortexed for 5 sec and centrifuged for 45 min at 11,000×g at 0-4° C.Decanting, resuspension in LiCl, and centrifugation were repeated. Thefinal pellet was air dried and resuspended in 2 ml of 1 mM EDTA, 0.5%SDS, 10 mM Tris (pH 7.5). Then, 20 μl of Proteinase K (20 mg/ml) wereadded, and the solution was incubated for 30 min at 37° C. withoccasional mixing. One-tenth volume (0.22-0.25 ml) of 3 M NaCl was addedand the solution was vortexed before transfer into another tube whichcontained 2 ml of phenol/chloroform/isoamyl alcohol (PCI). The tube wasvortexed for 1-3 sec and centrifuged for 20 min at 3,000×g at 10° C. ThePCI extraction was repeated twice more, followed by two similarextractions with chloroform/isoamyl alcohol. The final aqueous solutionwas transferred to a pre-chilled 15 ml corex glass tube containing 6 mlof 100% absolute ethanol, the tube was covered with parafilm and placedat −20° C. overnight. The tube was centrifuged for 30 min at 10,000×g at0-4° C., and the ethanol supernatant was decanted immediately. The RNApellet was washed four times with 10 ml of 75% ice-cold ethanol,followed each time by centrifugation at 10,000×g for 10 min. The finalpellet was air dried for 15 min at room temperature. The RNA wassuspended in 0.5 ml of 10 mM Tris (pH 7.6), 1 mM EDTA, and itsconcentration was determined spectrophotometrically. RNA samples werealiquoted and stored at −70° C. as ethanol precipitates.

The quality of the RNA was determined by agarose gel electrophoresis(see Example 5) and staining with 0.5 μg/ml ethidium bromide for onehour. RNA samples that did not contain intact 28S/18S rRNAs wereexcluded from the study.

Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent wasadded to 120 mg of pulverized tissue in a 2.0 ml polypropylene microfugetube, homogenized with a Polytrom® homogenizer (Brinkman Instruments,Inc., Westbury, N.Y.) for 50 sec and left on ice for 5 min. Then, 0.2 mlof chloroform was added to each sample, followed by vortexing for 15sec. The sample was left in ice for another 5 min, followed bycentrifugation at 12,000×g for 15 min at 4° C. The upper layer wascollected and transferred to another RNase-free 2.0 ml microfuge tube.An equal volume of isopropanol was added to each sample, and thesolution was placed on ice for 10 min. The sample was centrifuged at12,000×g for 10 min at 4° C., and the supernatant was discarded. Theremaining pellet was washed twice with cold 75% ethanol, resuspended byvortexing, and the resuspended material was then re-pelleted bycentrifugation at 7500×g for 5 min at 4° C. Finally, the RNA pellet wasdried in a speedvac for at least 5 min and reconstituted in RNase-freewater.

B. RNA Extraction from Blood Mononuclear Cells. Mononuclear cells areisolated from blood samples from patients by centrifugation usingFicoll-Hypaque as follows. A 10 ml volume of whole blood is mixed withan equal volume of RPMI Medium (Life Technologies, Gaithersburg, Md.).This mixture is then underlayed with 10 ml of Ficoll-Hypaque (Pharmacia,Piscataway, N.J.) and centrifuged for 30 minutes at 200×g. The buffycoat containing the mononuclear cells is removed, diluted to 50 ml withDulbecco's PBS (Life Technologies, Gaithersburg, Md.) and the mixturecentrifuged for 10 minutes at 200×g. After two washes, the resultingpellet is resuspended in Dulbecco's PBS to a final volume of 1 ml.

RNA is prepared from the isolated mononuclear cells as described by N.Kato et al., supra. Briefly, the pelleted mononuclear cells are broughtto a final volume of 1 ml and then are resuspended in 250 μL of PBS andmixed with 2.5 ml of 3M LiCl, 6M urea, 5 mM EDTA, 0.1M2-mercaptoethanol, 50 mM Tris-HCl (pH 7.5). The resulting mixture ishomogenized and incubated at −20° C. overnight. The homogenate is spunat 8,000 RPM in a Beckman J2-21M rotor for 90 minutes at 0-4° C. Thepellet is resuspended in 10 ml 3M LiCl by vortexing and then spun at10,000 RPM in a Beckman J2-21M rotor centrifuge for 45 minutes at 0-4°C. The resuspending and pelleting steps then are repeated. The pellet isresuspended in 2 ml of 1 mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5) and 400μg Proteinase K with vortexing and then it is incubated at 37° C. for 30minutes with shaking. One tenth volume of 3M NaCl then is added and thevortexed mixture. Proteins are removed by two cycles of extraction withphenol/chloroform/isoamyl alcohol followed by one extraction withchloroform/isoamyl alcohol. RNA is precipitated by the addition of 6 mlof ethanol followed by overnight incubation at −20° C. After theprecipitated RNA is collected by centrifugation, the pellet is washed 4times in 75% ethanol. The pelleted RNA is then dissolved in 1 mM EDTA,10 mM Tris-HCl (pH 7.5).

Non-breast tissues are used as negative controls. The mRNA can befurther purified from total RNA by using commercially available kitssuch as oligo dT cellulose spin columns (RediCol™ from Pharmacia,Uppsala, Sweden) for the isolation of poly-adenylated RNA. Total or mRNAcan be dissolved in lysis buffer (5M guanidine thiocyanate, 0.1M EDTA,pH 7.0) for analysis in the ribonuclease protection assay.

C. RNA Extraction from polysomes. Tissue is minced in saline at 4° C.and mixed with 2.5 volumes of 0.8 M sucrose in a TK₁₅₀M (150 mM KCl, 5mM MgCl₂, 50 mM Tris-HCl, pH 7.4) solution containing 6 mM2-mercaptoethanol. The tissue is homogenized in a Teflon-glass Potterhomogenizer with five strokes at 100-200 rpm followed by six strokes ina Dounce homogenizer, as described by B. Mechler, Methods in Enzymology152:241-248 (1987). The homogenate then is centrifuged at 12,000×g for15 min at 4° C. to sediment the nuclei. The polysomes are isolated bymixing 2 ml of the supernatant with 6 ml of 2.5 M sucrose in TK₁₅₀M andlayering this mixture over 4 ml of 2.5 M sucrose in TK₁₅₀M in a 38 mlpolyallomer tube. Two additional sucrose TK₁₅₀M solutions aresuccessively layered onto the extract fraction; a first layer of 13 ml2.05 M sucrose followed by a second layer of 6 ml of 1.3 M sucrose. Thepolysomes are isolated by centrifuging the gradient at 90,000×g for 5 hat 4° C. The fraction then is taken from the 1.3 M sucrose/2.05 Msucrose interface with a siliconized pasteur pipette and diluted in anequal volume of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). An equal volumeof 90° C. SDS buffer (1% SDS, 200 mM NaCl, 20 mM Tris-HCl, pH 7.4) isadded and the solution is incubated in a boiling water bath for 2 min.Proteins next are digested with a Proteinase-K digestion (50 mg/ml) for15 min at 37° C. The mRNA is purified with 3 equal volumes ofphenol-chloroforn extractions followed by precipitation with 0.1 volumeof 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at −20° C.overnight. The precipitated RNA is recovered by centrifugation at12,000×g for 10 min at 4° C. The RNA is dried and resuspended in TE (pH7.4) or distilled water. The resuspended RNA then can be used in a slotblot or dot blot hybridization assay to check for the presence of BU101mRNA (see Example 6).

The quality of nucleic acid and proteins is dependent on the method ofpreparation used. Each sample may require a different preparationtechnique to maximize isolation efficiency of the target molecule. Thesepreparation techniques are within the skill of the ordinary artisan.

Example 4 Ribonuclease Protection Assay

A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization Probe andUnlabeled Sense Strand. A pSPORT1 plasmid containing the BU101 gene cDNAsequence insert (clone 603148), flanked by opposed SP6 and T7 polymerasepromoters, was purified using Qiagen Plasmid Purification Kit (Qiagen,Chatsworth, Calif.). Then, 10 μg of the plasmid were cut with 10 U Dde Irestriction enzyme for 1 h at 37° C. The cut plasmid was purified usingQIAprep kits (Qiagen, Chatsworth, Calif.) and used for the synthesis ofantisense transcript labeled with 6.3 μM (alpha³²P) UTP (Amersham LifeSciences, Inc. Arlington Heights, Ill.) from the SP6 promoter using theRiboprobe® in vitro Transcription System (Promega Corporation, Madison,Wis.), as described by the supplier's instructions. To generate thesense strand, 10 μg of the purified plasmid were cut with restrictionenzymes 10U Xba I and 10 U Not I, and transcribed as above from the T7promoter. Both sense and antisense strands were isolated by spin columnchromatography. Unlabeled sense strand was quantitated by UV absorptionat 260 nm.

B. Hybridization of Labeled Probe to Target. Frozen tissue waspulverized to powder under liquid nitrogen and 100-500 mg were dissolvedin 1 ml of lysis buffer as available as a component of theDirectProtect™ Lysate RNase Protection kit (Ambion, Inc., Austin, Tex.).Further dissolution was achieved using a tissue homogenizer. Inaddition, a dilution series of a known amount of sense strand in mouseliver lysate was made for use as a positive control. Finally, 45 μl ofsolubilized tissue or diluted sense strand was mixed directly with 1×10⁵cpm of radioactively labeled probe in 5 μl of lysis buffer.Hybridization was allowed to proceed overnight at 37° C.

C. RNase Digestion. RNA that was not hybridized to probe was removedfrom the reaction as per the Direct Protect™ protocol using a solutionof RNase A and RNase T1 for 30 min at 37° C., followed by removal ofRNase by Proteinase-K digestion in the presence of sodium sarcosyl.Hybridized fragments protected from digestion were then precipitated bythe addition of an equal volume of isopropanol and placed at −70° C. for3 h. The precipitates were collected by centrifugation at 12,000×g for20 min.

D. Fragment Analysis. The precipitates were dissolved in denaturing gelloading dye (80% formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol,1 mg/ml bromophenol blue), heat denatured, and electrophoresed in 6%polyacrylamide TBE, 8 M urea denaturing gels. The gels were imaged andanalyzed using the STORM™ storage phosphor autoradiography system(Molecular Dynamics, Sunnyvale, Calif.). Quantitation of protectedfragment bands, expressed in femtograms (fg), was achieved by comparingthe peak areas obtained from the test samples to those from the knowndilutions of the positive control sense strand (see Section B, supra).In addition, the concentration of DNA in the lysate was assayed toestimate the number of cells in the test sample lysates. The results areexpressed in molecules of BU101 RNA/cell and as a image rating score(Table 1). High level expression of mRNA corresponding to a sequenceselected from the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments or complementsthereof, indicated the presence of BU101 mRNA(s), suggesting a diagnosisof a breast tissue disease or condition, such as breast cancer.

TABLE 1 Tissue ID Number BU101 RNA/cell Score* Normal Breast C157 10 +C007G 7 + C027R 8 + C016R 2 + C135R 0.2 + Malignant Breast C011G >46 3+C023G 0.3 + C012G 0 − C033R >87 3+ C030 0 − Normal Lung C005R 0 −Malignant Lung C037G 0 − Normal Colon C027G 0 − *Samples with nodetectable protected fragment were scored “−”; samples with detectableprotected fragment, the fg values of which were within the std curve,were scored “+”; samples with detectable protected fragment, the fgvalues of which were 2 to 10 fold above the std curve, were scored “2+”;samples with detectable protected fragment, the fg values of which were10 fold or more above the std curve, were scored “3+”

Example 5 Northern Blotting

The northern blot technique was used to identify a specific size RNAspecies in a complex population of RNA using agarose gel electrophoresisand nucleic acid hybridization. Briefly, 5-10 μg of total RNA (seeExample 3) was incubated in 15 μl of a solution containing 40 mMmorphilinopropanesulfonic acid (MOPS) (pH 7.0), 10 mM sodium acetate, 1mM EDTA, 2.2 M formaldehyde, 50% v/v formamide for 15 min at 65° C. Thedenatured RNA was mixed with 2 μl of loading buffer (50% glycerol, 1 mMEDTA, 0.4% bromophenol blue, 0.4% xylene cyanol) and loaded into adenaturing 1.0% agarose gel containing 40 mM MOPS (pH 7.0), 10 mM sodiumacetate, 1 mM EDTA and 2.2 M formaldehyde. The gel was electrophoresedat 60 V for 1.5 h and rinsed in RNAse free water. Gels were stained with0.5 μg/ml of ethidium bromide in RNAse free water and illuminated withUV light to visualize ribosomal RNA bands. RNA was transferred from thegel onto nylon membranes (Brightstar-Plus, Ambion, Inc., Austin, Tex.)for 1.5 hours using the downward alkaline capillary transfer method(Chomczynski, Anal. Biochem. 201:134-139, 1992). The filter was rinsedwith 1×SSC, and RNA was crosslinked to the filter using a Stratalinker(Stratagene, Inc., La Jolla, Calif.) on the autocrosslinking mode anddried for 15 min. The membrane was then placed into a hybridization tubecontaining 20 ml of preheated prehybridization solution (5×SSC, 50%formamide, 5×Denhardt's solution, 100 μg/ml denatured salmon sperm DNA)and incubated in a 42° C. hybridization oven for at least 3 hr. Whilethe blot was prehybridizing, a ³²P-labeled random-primed probe wasgenerated using the BU101 insert fragment (obtained by digesting clone603148 with XbaI and NotI) using Random Primer DNA Labeling System (LifeTechnologies, Inc., Gaithersburg, Md.) according to the manufacturer'sinstructions. Half of the probe was boiled for 10 min, quick chilled onice and added to the hybridization tube. Hybridization was carried outat 42° C. for at least 12 hr. The hybridization solution was discardedand the filter was washed in 30 ml of 3×SSC, 0.1 % SDS at 42° C. for 15min, followed by 30 ml of 3×SSC, 0.1 % SDS at 42° C. for 15 min. Thefilter was wrapped in saran wrap, exposed to Kodak XAR-Omat film for8-96 hr, and the film was developed for analysis.

Results of the analysis of RNA quality using an ethidium bromide stainedagarose gel and the corresponding northern blot using BU101 probehybridized to RNAs from breast tissues and non-breast tissues are shownin FIGS. 3A & B, respectively. The positions of RNA size standards (inkb) are shown to the left of each panel. The BU101 probe hybridized toan RNA band at 0.5 kb only in a breast sample in lane 1 but not to RNAsin the breast sample in lane 3 or the seven non-breast samples in lanes4-10 (colon, colon, lung, lung, ovary, prostate, and spleen,respectively) (FIG. 3B). Lane 2 is blank.

Example 6 Dot Blot/Slot Blot

Dot and slot blot assays are quick methods to evaluate the presence of aspecific nucleic acid sequence in a complex mix of nucleic acid. Toperform such assays, up to 50 μg of RNA is mixed in 50 μl of 50%formamide, 7% formaldehyde, 1×SSC, incubated 15 min at 68° C., and thencooled on ice. Then, 100 μl of 20×SSC is added to the RNA mixture andloaded under vacuum onto a manifold apparatus that has a preparednitrocellulose or nylon membrane. The membrane is soaked in water,20×SSC for 1 hour, placed on two sheets of 20×SSC prewet Whatman #3filter paper, and loaded into a slot blot or dot blot vacuum manifoldapparatus. The slot blot is analyzed with probes prepared and labeled asdescribed in Example 4, supra. Detection of mRNA corresponding to asequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof, is an indication of the presence of BU101,suggesting a diagnosis of a breast tissue disease or condition, such asbreast cancer.

Other methods and buffers which can be utilized in the methods describedin Examples 5 and 6, but not specifically detailed herein, are known inthe art and are described in J. Sambrook et al, supra which isincorporated herein by reference.

Example 7 In Situ Hybridization

This method is useful to directly detect specific target nucleic acidsequences in cells using detectable nucleic acid hybridization probes.

Tissues are prepared with cross-linking fixative agents such asparaformaldehyde or glutaraldehyde for maximum cellular RNA retention.See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991). Briefly,the tissue is placed in greater than 5 volumes of 1% glutaraldehyde in50 mM sodium phosphate, pH 7.5 at 4° C. for 30 min. The solution ischanged with fresh glutaraldehyde solution (1% glutaraldehyde in 50 mMsodium phosphate, pH 7.5) for a further 30 min fixing. The fixingsolution should have an osmolality of approximately 0.375% NaCl. Thetissue is washed once in isotonic NaCl to remove the phosphate.

The fixed tissues then are embedded in paraffin as follows. The tissueis dehydrated though a series of ethanol concentrations for 15 min each:50% (twice), 70% (twice), 85%, 90% and then 100% (twice). Next, thetissue is soaked in two changes of xylene for 20 min each at roomtemperature. The tissue is then soaked in two changes of a 1:1 mixtureof xylene and paraffin for 20 min each at 60° C.; and then in threefinal changes of paraffin for 15 min each.

Next, the tissue is cut in 5 μm sections using a standard microtome andplaced on a slide previously treated with a tissue adhesive such as3-aminopropyltriethoxysilane.

Paraffin is removed from the tissue by two 10 min xylene soaks andrehydrated in a series of ethanol concentrations: 99% twice, 95%, 85%,70%, 50%, 30%, and then distilled water twice. The sections arepre-treated with 0.2 M HCl for 10 min and permeabilized with 2 μg/mlProteinase-K at 37° C. for 15 min.

Labeled riboprobes transcribed from the BU101 gene plasmid (see Example4) are hybridized to the prepared tissue sections and incubatedovernight at 56° C. in 3×standard saline extract and 50% formamide.Excess probe is removed by washing in 2×standard saline citrate and 50%formamide followed by digestion with 100 μg/ml RNase A at 37° C. for 30min. Fluorescence probe is visualized by illumination with ultraviolet(UV) light under a microscope. Fluorescence in the cytoplasm isindicative of BU101 mRNA. Alternatively, the sections can be visualizedby autoradiography.

Example 8 Reverse Transcription PCR

A. One Step RT-PCR Assay. Target-specific primers are designed to detectthe above-described target sequences by reverse transcription PCR usingmethods known in the art. One step RT-PCR is a sequential procedure thatperforms both RT and PCR in a single reaction mixture. The procedure isperformed in a 200 μl reaction mixture containing 50 mM(N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH,0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraaceticacid, 0.02 mg/ml NaN₃, 8% w/v glycerol, 150 μM each of dNTP, 0.25 μMeach primer, 5U rTth polymerase, 3.25 mM Mn(OAc)₂ and 5 μl of target RNA(see Example 3). Since RNA and the rTth polymerase enzyme are unstablein the presence of Mn(OAc)₂, the Mn(OAc)₂ should be added just beforetarget addition. Optimal conditions for cDNA synthesis and thermalcycling readily can be determined by those skilled in the art. Thereaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimalconditions for cDNA synthesis and thermal cycling can readily bedetermined by those skilled in the art. Conditions which may be founduseful include cDNA synthesis at 60°-70° C. for 15-45 min and 30-45amplification cycles at 94° C., 1 min; 55°-70° C., 1 min; 72° C., 2 min.One step RT-PCR also may be performed by using a dual enzyme procedurewith Taq polymerase and a reverse transcriptase enzyme, such as MMLV orAMV RT enzymes.

B. Traditional RT-PCR. A traditional two-step RT-PCR reaction wasperformed, as described by K. Q. Hu et al., Virology 181:721-726 (1991).Briefly, 0.5 μg of extracted mRNA (see Example 3) was reversetranscribed in a 20 μl reaction mixture containing 1×PCR II buffer(Perkin-Elmer), 5 mM MgCl₂, 1 mM dNTP, 20 U RNasin, 2.5 μM randomhexamers, and 50 U MMLV (Moloney murine leukemia virus) reversetranscriptase (RT). Reverse transcription was performed at roomtemperature for 10 min, 42° C. for 60 min in a PE-480 thermal cycler,followed by further incubation at 95° C. for 5 min to inactivate the RT.PCR was performed using 2 μl of the cDNA reaction in a final PCRreaction volume of 50 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl,2 mM MgCl₂, 200 μM dNTP, 0.5 μLM of each sense and antisense primer,SEQUENCE ID NO 11 and SEQUENCE ID NO 12, respectively, and 2.5 U of Taqpolymerase. The reaction was incubated in an MJ Research Model PTC-200as follows: 40 cycles of amplification (94° C., 20 sec; 58° C., 30 sec;72° C., 30 sec); a final extension (72° C. 10 min); and a soak at 4° C.

C. PCR Fragment Analysis. The correct products were verified by sizedetermination using gel electrophoresis with a SYBR® Green I fluorescentintercalator (Molecular Probes, Eugene, Oreg.) and imaged using a STORMimaging system (FIG. 4). FIG. 4 shows a DNA band at 201 bases which isindicative of a BU101 specific PCR product, in both normal (lanes 1-5)and cancerous (lanes 6-10) breast tissues, and not in any lung (lanes12-16) or colon (lanes 17-21) tissues. Detection of a product comprisinga sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof, indicated the presence of BU101 mRNA(s), suggestinga diagnosis of a breast tissue disease or condition, such as breastcancer.

Example 9 OH-PCR

A. Probe selection and Labeling. Target-specific primers and probes aredesigned to detect the above-described target sequences byoligonucleotide hybridization PCR. International Publication Nos WO92/10505, published Jun. 25 1992, and WO 92/11388, published Jul. 9,1992, teach methods for labeling oligonucleotides at their 5′ and 3′ends, respectively. According to one known method for labeling anoligonucleotide, a label-phosphoramidite reagent is prepared and used toadd the label to the oligonucleotide during its synthesis. For example,see N. T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J. S.Cohen et al., published U.S. patent application Ser. No. 07/246,688(NTIS ORDER No. PAT-APPL-7-246,688) (1989). Preferably, probes arelabeled at their 3′ end to prevent participation in PCR and theformation of undesired extension products. For one step OH-PCR, theprobe should have a T_(M) at least 15° C. below the T_(M) of theprimers. The primers and probes are utilized as specific bindingmembers, with or without detectable labels, using standardphosphoramidite chemistry and/or post-synthetic labeling methods whichare well-known to one skilled in the art.

B. One Step Oligo Hybridization PCR. OH-PCR is performed on a 200 μlreaction containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15,81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mMethylene diaminetetraacetic acid, 0.02 mg/ml NaN₃, 8% w/v glycerol, 150μM each of dNTP, 0.25 μM each primer, 3.75 nM probe, 5U rTth polymerase,3.25 mM Mn(OAc)₂ and 5 μl blood equivalents of target (see Example 3).Since RNA and the rTth polymerase enzyme are unstable in the presence ofMn(OAc)₂, the Mn(OAc)₂ should be added just before target addition. Thereaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimalconditions for cDNA synthesis and thermal cycling can be readilydetermined by those skilled in the art. Conditions which may be founduseful include cDNA synthesis (60° C., 30 min), 30-45 amplificationcycles (94° C., 40 sec; 55-70° C., 60 sec), oligo-hybridization (97° C.,5 min; 15° C. 5 min; 15° C. soak). The correct reaction product containsat least one of the strands of the PCR product and an internallyhybridized probe.

C. OH-PCR Product Analysis. Amplified reaction products are detected onan LCx® analyzer system (available from Abbott Laboratories, AbbottPark, Ill.). Briefly, the correct reaction product is captured by anantibody labeled microparticle at a capturable site on either the PCRproduct strand or the hybridization probe, and the complex is detectedby binding of a detectable antibody conjugate to either a detectablesite on the probe or the PCR strand. Only a complex containing a PCRstrand hybridized with the internal probe is detectable. The detectionof this complex then is indicative of the presence of BU101 mRNA,suggesting a diagnosis of a breast disease or condition, such as breastcancer.

Many other detection formats exist which can be used and/or modified bythose skilled in the art to detect the presence of amplified ornon-amplified BU101-derived nucleic acid sequences including, but notlimited to, ligase chain reaction (LCR, Abbott Laboratories, AbbottPark, Ill.); Q-beta replicase (Gene-Trak™, Naperville, Ill.), branchedchain reaction (Chiron, Emeryville, Calif.) and strand displacementassays (Becton Dickinson, Research Triangle Park, N.C.).

Example 10 Synthetic Peptide Production

Synthetic peptides, BU101.1-BU101.8 (SEQ ID NOS 16-23, respectively)were prepared based upon the predicted amino acid sequence of the openreading frame of BU101 (SEQUENCE ID NO 15) (see Example 1). All peptideswere synthesized on a Symphony Peptide Synthesizer (available fromRainin Instrument Co, Emeryville Calif.), using FMOC chemistry, standardcycles and in-situ HBTU activation. Cleavage and deprotection conditionswere as follows: a volume of 2.5 ml of cleavage reagent (77.5% v/vtrifluoroacetic acid, 15% v/v ethanedithiol, 2.5% v/v water, 5% v/vthioanisole, 1-2% w/v phenol) was added to the resin, and agitated atroom temperature for 2-4 hours. The filtrate was then removed and thepeptide was precipitated from the cleavage reagent with cold diethylether. Each peptide was then filtered, purified via reverse-phasepreparative HPLC using a water/acetonitrile/0.1% TFA gradient, andlyophilized. The product was confirmed by mass spectrometry (data notshown).

The purified peptides were conjugated to Keyhole Limpet Hemocyanin withglutaraldehyde, mixed with adjuvant, and injected into rabbits (seeExample 14).

Example 11a Expression of Protein in a Cell Line Using Plasmid 577

A. Construction of a BU101 Expression Plasmid. Plasmid 577, described inU.S. patent application Ser. No. 08/478,073, filed Jun. 7, 1995, nowU.S. Pat. No. 6,020,122, and incorporated herein by reference, has beenconstructed for the expression of secreted antigens in a permanent cellline. This plasmid contains the following DNA segments: (a) a 2.3 Kbfragment of pBR322 containing bacterial beta-lactamase and origin of DNAreplication; (b) a 1.8 Kb cassette directing expression of a neomycinresistance gene under control of HSV-1 thymidine kinase promoter andpoly-A addition signals; (c) a 1.9 Kb cassette directing expression of adihydrofolate reductase gene under the control of an SV-40 promoter andpoly-A addition signals; (d) a 3.5 Kb cassette directing expression of arabbit immunoglobulin heavy chain signal sequence fused to a modifiedhepatitis C virus (HCV) E2 protein under the control of the Simian Virus40 T-Ag promoter and transcription enhancer, the hepatitis B virussurface antigen (HBsAg) enhancer I followed by a fragment of HerpesSimplex Virus-1 (HSV-1) genome providing poly-A addition signals; and(e) a residual 0.7 Kb fragment of Simian Virus 40 genome late region ofno function in this plasmid. All of the segments of the vector wereassembled by standard methods known to those skilled in the art ofmolecular biology.

Plasmids for the expression of secretable BU101 proteins are constructedby replacing the hepatitis C virus E2 protein coding sequence in plasmid577 with that of a BU101 polynucleotide sequence selected from the groupconsisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,SEQUENCE ID NO 4, and fragments or complements thereof, as follows.Digestion of plasmid 577 with XbaI releases the hepatitis C virus E2gene fragment. The resulting plasmid backbone allows insertion of theBU101 cDNA insert downstream of the rabbit immunoglobulin heavy chainsignal sequence which directs the expressed proteins into the secretorypathway of the cell. The BU101 cDNA fragment is generated by PCR usingstandard procedures. Encoded in the sense PCR primer sequence is an XbaIsite, immediately followed by a 12 nucleotide sequence that encodes theamino acid sequence Ser-Asn-Glu-Leu (“SNEL”) to promote signal proteaseprocessing, efficient secretion and final product stability in culturefluids. Immediately following this 12 nucleotide sequence the primercontains nucleotides complementary to template sequences encoding aminoacids of the BU101 gene. The antisense primer incorporates a sequenceencoding the following eight amino acids just before the stop codons:Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE ID NO 24). Within thissequence is incorporated a recognition site to aid in analysis andpurification of the BU101 protein product. A recognition site (termed“FLAG”) that is recognized by a commercially available monoclonalantibody designated anti-FLAG M2 (Eastman Kodak, Co., New Haven, Conn.)can be utilized, as well as other comparable sequences and theircorresponding antibodies. For example, PCR is performed using GeneAmp®reagents obtained from Perkin-Elmer-Cetus, as directed by the supplier'sinstructions. PCR primers are used at a final concentration of 0.5 μM.PCR is performed on the BU101 plasmid template in a 100 μl reaction for35 cycles (94° C., 30 seconds; 55° C., 30 seconds; 72° C., 90 seconds)followed by an extension cycle of 72° C. for 10 min.

B. Transfection of Dihydrofolate Reductase Deficient Chinese HamsterOvary Cells. The plasmid described supra is transfected intoCHO/dhfr-cells (DXB-111, Uriacio, et al., PNAS 77:4451-4466 (1980)).These cells are available from the A.T.C.C., 12301 Parklawn Drive,Rockville, Md. 20852, under Accession No. CRL 9096. Transfection iscarried out using the cationic liposome-mediated procedure described byP. L. Felgner et al., PNAS 84:7413-7417 (1987). Particularly,CHO/dhfr-cells are cultured in Ham's F-12 media supplemented with 10%fetal calf serum, L-glutamine (1 mM) and freshly seeded into a flask ata density of 5-8×10⁵ cells per flask. The cells are grown to aconfluency of between 60 and 80% for transfection. Twenty micrograms (20μg) of plasmid DNA is added to 1.5 ml of Opti-MEM I medium and 100 gl ofLipofectin Reagent (Gibco-BRL; Grand Island, N.Y.) are added to a second1.5 ml portion of Opti-MEM I media. The two solutions are mixed andincubated at room temperature for 20 min. After the culture medium isremoved from the cells, the cells are rinsed 3 times with 5 ml ofOpti-MEM I medium. The Opti-MEM I-Lipofection-plasmid DNA solution thenis overlaid onto the cells. The cells are incubated for 3 h at 37° C.,after which time the Opti-MEM I-Lipofectin-DNA solution is replaced withculture medium for an additional 24 h prior to selection.

C. Selection and Amplification. One day after transfection, cells arepassaged 1:3 and incubated with dhfr/G418 selection medium (hereafter,“F-12 minus medium G”). Selection medium is Ham's F-12 with L-glutamineand without hypoxanthine, thymidine and glycine (JRH Biosciences,Lenexa, Kans.) and 300 μg per ml G418 (Gibco-BRL; Grand Island, N.Y.).Media volume-to-surface area ratios of 5 ml per 25 cm² are maintained.After approximately two weeks, DHFR/G418 cells are expanded to allowpassage and continuous maintenance in F-12 minus medium G.

Amplification of each of the transfected BU101 cDNA sequences isachieved by stepwise selection of DHFR⁺, G418⁺ cells with methotrexate(reviewed by R. Schimke, Cell 37:705-713 (1984)). Cells are incubatedwith F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma,St. Louis, Mo.) for approximately two weeks until resistant coloniesappear. Further gene amplification is achieved by selection of 150 nMadapted cells with 5 μM MTX.

D. Antigen Production. F-12 minus medium G supplemented with 5 μM MTX isoverlaid onto just confluent monolayers for 12 to 24 h at 37° C. in 5%CO₂. The growth medium is removed and the cells are rinsed 3 times withDulbecco's phosphate buffered saline (PBS) (with calcium and magnesium)(Gibco-BRL; Grand Island, N.Y.) to remove the remaining media/serumwhich may be present. Cells then are incubated with VAS custom medium(VAS custom formulation with L-glutamine with HEPES without phenol red,available from JRH Bioscience; Lenexa, Kans., product number 52-08678P),for 1 h at 37° C. in 5% CO₂. Cells then are overlaid with VAS forproduction at 5 ml per T flask. Medium is removed after seven days ofincubation, retained, and then frozen to await purification withharvests 2, 3 and 4. The monolayers are overlaid with VAS for 3 moreseven day harvests.

E. Analysis of Breast Tissue Gene BU101 Antigen Expression. Aliquots ofVAS supernatants from the cells expressing the BU101 protein constructare analyzed, either by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using standard methods and reagents known in the art (Laemmlidiscontinuous gels), or by mass spectrometry.

F. Purification. Purification of the BU101 protein containing the FLAGsequence is performed by immunoaffinity chromatography using an affinitymatrix comprising anti-FLAG M2 monoclonal antibody covalently attachedto agarose by hydrazide linkage (Eastman Kodak Co., New Haven, Conn.).Prior to affinity purification, protein in pooled VAS medium harvestsfrom roller bottles is exchanged into 50 mM Tris-HCl (pH 7.5), 150 mMNaCl buffer using a Sephadex G-25 (Pharmacia Biotech Inc., Uppsala,Sweden) column. Protein in this buffer is applied to the anti-FLAG M2antibody affinity column. Non-binding protein is eluted by washing thecolumn with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer. Bound proteinis eluted using an excess of FLAG peptide in 50 mM Tris-HCl (pH 7.5),150 mM NaCl. The excess FLAG peptide can be removed from the purifiedBU101 protein by gel electrophoresis or HPLC.

Although plasmid 577 is utilized in this example, it is known to thoseskilled in the art that other comparable expression systems, such asCMV, can be utilized herein with appropriate modifications in reagentand/or techniques and are within the skill of the ordinary artisan.

The largest cloned insert containing the coding region of the BU101 geneis sub-cloned into either (i) a eukaryotic expression vector which maycontain, for example, a cytomegalovirus (CMV) promoter and/or proteinfusible sequences which aid in protein expression and detection, or (ii)a bacterial expression vector containing a superoxide-dismutase (SOD)and CMP-KDO synthetase (CKS) or other protein fusion gene for expressionof the protein sequence. Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in EPO 0196056, published Oct. 1, 1986, which is incorporatedherein by reference and those containing fusion sequences of CKS aredescribed in EPO Publication No. 0331961, published Sep. 13, 1989, whichpublication is also incorporated herein by reference. This so-purifiedprotein can be used in a variety of techniques, including but notlimited to animal immunization studies, solid phase immunoassays, etc.

Example 11b Expression of Protein in a Cell Line Using pcDNA3.1/Myc-His

A. Construction of a BU101 Expression Plasmid. Plasmid pcDNA3.1/Myc-His(Cat.# V855-20, Invitrogen, Carlsbad, Calif.) has been constructed, inthe past, for the expression of secreted antigens by most mammalian celllines. Expressed protein inserts are fused to a myc-his peptide tag. Themyc-his tag (SEQUENCE ID NO 25) comprises a c-myc oncoprotein epitopeand a polyhistidine sequence which are useful for the purification of anexpressed fusion protein by using either anti-myc or anti-his affinitycolumns, or metalloprotein binding columns.

A plasmid for the expression of secretable BU101 protein was constructedby inserting a BU101 polynucleotide sequence from clone 603148 into thepcDNA3.1/Myc-His vector. Prior to construction of a BU101 expressionplasmid, the BU101 cDNA sequence was first cloned into a pCR®-Bluntvector. The BU101 cDNA fragment was generated by PCR performed usingStratagene® reagents obtained from Stratagene, as directed by thesupplier's instructions. PCR primers are used at a final concentrationof 0.5 μM. PCR using 5 U of pfu polymerase (Stratagene, La Jolla,Calif.) is performed on the BU101 plasmid template (see Example 2) in a50 μl reaction for 30 cycles (94° C., 1 min; 65° C., 1.5 min; 72° C., 3min) followed by an extension cycle of 72° C. for 8 min. (The sense PCRprimer sequence, SEQUENCE ID NO 13, comprises nucleotides which areidentical to the pSPORT vector directly upstream of the BU101 geneinsert. The antisense primer, SEQUENCE ID NO 14, incorporated a 5′ Not Irestriction sequence and a sequence complementary to the 3′ end of theBU101 cDNA insert just upstream of the 3′-most, in-frame stop codon.)Five microliters (5 μl) of the resulting blunted-ended PCR product wereligated into 25 ng of linearized pCR®-Blunt vector (Invitrogen,Carlsbad, Calif.) interrupting the lethal ccdB gene of the vector. Theresulting ligated vector was transformed into TOP 10 E. coli(Invitrogen, Carlsbad, Calif.) using a One Shot™ transformation kit(Invitrogen, Carlsbad, Calif.) following supplier's directions. Thetransformed cells were grown on LB-Kan (50 μg/ml kanamycin) selectionplates at 37° C. Only cells containing a plasmid with an interruptedccdB gene grew after transformation (Grant, S. G. N., PNAS 87:4645-4649(1990)). Transformed colonies were picked and grown up in 3 ml of LB-Kanbroth at 37° C. Plasmid DNA was isolated by using a QIAprep® (QiagenInc., Santa Clarita, Calif.) procedure, as directed by the supplier'sinstructions. The DNA was digested with EcoRI and NotI restrictionenzymes to release the BU101 insert fragment. The fragment waselectrophoresed on 1% Seakem® LE agarose (FMC, Rockland, Me.)/0.5 μg/mlethidium bromide/TE gel, visualized by UV illumination, excised andpurified using QIAquick™ (Qiagen Inc., Santa Clarita, Calif.)procedures, as directed by the supplier's instructions.

The pcDNA3. 1/Myc-His plasmid DNA was linearized by digestion with EcoRIand NotI, sites present in the polylinker region of the plasmid DNA. TheBU101 purified fragment, supra, was ligated with the resulting plasmidDNA backbone downstream from a CMV promoter, and transformed into DH5alpha™ cells (GibcoBRL Gaithersburg, Md.), as directed by the supplier'sinstructions. Briefly, 10 ng of pcDNA3.1/Myc-His containing the BU101insert were added to 50 μl of competent DH5 alpha cells, and thecontents were mixed gently. The mixture was incubated on ice for 30 min,heat shocked for 20 sec at 37° C., and placed on ice for an additional 2min. Upon addition of 0.95 ml of LB medium, the mixture was incubatedfor 1 h at 37° C. while shaking at 225 rpm. The transformed cells thenwere plated onto 100 mm LB/Amp (50 μg/ml ampicillin) plates and grown at37° C. Colonies were picked and grown in 3 ml of LB/ampicillin broth.Plasmid DNA was purified using a QIAprep kit. The presence of the insertwas confirmed using restriction enzyme digestion and gel analysis. (J.Sambrook et al., supra.)

B. Transfection of Human Embryonic Kidney Cell 293 Cells. The BU101expression plasmid described in section A, supra, was retransformed intoDH5 alpha cells, plated onto LB/ampicillin agar, and grown up in 10 mlof LB/ampicillin broth, as described hereinabove. The plasmid waspurified using a QIAfilter™ Maxi kit (Qiagen, Chatsworth, Calif.) andtransfected into HEK293 cells (F. L. Graham et al., J. Gen. Vir.36:59-72 (1977)). These cells are available from the A.T.C.C., 12301Parklawn Drive, Rockville, Md. 20852, under Accession No. CRL 1573.Transfection was carried out using the cationic lipofectamine-mediatedprocedure described by P. Hawley-Nelson et al., Focus 15.73 (1993).HEK293 cells were cultured in 10 ml DMEM media supplemented with 10%fetal bovine serum (FBS), L-glutamine (2 mM) and freshly seeded into 100mm culture plates at a density of 9×10⁶ cells per plate. The cells weregrown at 37° C. to a confluency of between 70% and 80% for transfection.Eight micrograms (8 μg) of plasmid DNA were added to 800 μl of Opti-MEMI® medium (Gibco-BRL, Grand Island, N.Y.), and 48-96 μl ofLipofectamine™ Reagent (Gibco-BRL, Grand Island, N.Y.) were added to asecond 800 μl portion of DMEM serum-free medium. The two solutions weremixed and incubated at room temperature for 15-30 min. After the culturemedium was removed from the cells, the cells were washed once with 10 mlof serum-free DMEM. The Opti-MEM I-Lipofectamine-plasmid DNA solutionwas diluted with 6.4 ml of serum-free DMEM and then overlaid onto thecells. The cells were incubated for 5 h at 37° C., after which time, anadditional 8 ml of DMEM with 20% FBS were added. After 18-24 h, the oldmedium was aspirated, and the cells were overlaid with 5 ml of freshDMEM with 5% FBS. Supernatants and cell extracts were analyzed for BU101gene activity 72 h after transfection.

C. Analysis of Breast Tissue Gene BU101 Antigen Expression. The culturesupernatant, supra, is transferred to cryotubes and stored on ice.HEK293 cells are harvested by washing twice with 10 ml of coldDulbecco's PBS and lysing by addition of 1.5 ml of CAT lysis buffer(Boehringer Mannheim, Indianapolis, Ind.), followed by incubation for 30min at room temperature. Lysate is transferred to 1.7 ml polypropylenemicrofuge tubes and centrifuged at 1000×g for 10 min. The supernatant istransferred to new cryotubes and stored on ice. Aliquots of supernatantsfrom the cells and the lysate of the cells expressing the BU101 proteinconstruct are analyzed for the presence of BU101 recombinant protein.The aliquots can be run on SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using standard methods and reagents known in the art. (J.Sambrook et al., supra) These gels can then be blotted onto a solidmedium such as nitrocellulose, nytran, etc., and the BU101 protein bandcan be visualized using western blotting techniques with anti-mycepitope or anti-histidine monoclonal antibodies (Invitrogen, Carlsbad,Calif.) or anti-BU101 polyclonal serum (see Example 14). Alternatively,the expressed BU101 recombinant protein can be analyzed by massspectrometry (see Example 12).

D. Purification. Purification of the BU101 recombinant proteincontaining the myc-his sequence is performed using the Xpress® affinitychromatography system (Invitrogen, Carlsbad, Calif.) containing anickel-charged agarose resin which specifically binds polyhistidineresidues. Supernatants from 10×100 mm plates, prepared as describedsupra, are pooled and passed over the nickel-charged column. Non-bindingprotein is eluted by washing the column with 50 mM Tris-HCl (pH 7.5)/150mM NaCl buffer, leaving only the myc-his fusion proteins. Bound BU101recombinant protein then is eluted from the column using either anexcess of imidazole or histidine, or a low pH buffer. Alternatively, therecombinant protein can also be purified by binding at the myc-hissequence to an affinity column consisting of either anti-myc oranti-histidine monoclonal antibodies conjugated through a hydrazide orother linkage to an agarose resin and eluting with an excess of mycpeptide or histidine, respectively.

The purified recombinant protein can then be covalently cross-linked toa solid phase, such as N-hydroxysuccinimide-activated sepharose columns(Pharmacia Biotech, Piscataway, N.J.), as directed by supplier'sinstructions. These columns containing covalently linked BU101recombinant protein, can then be used to purify anti-BU101 antibodiesfrom rabbit or mouse sera (see Examples 13 and 14).

E. Coating Microtiter Plates with BU101 Expressed Proteins. Supernatantfrom a 100 mm plate, as described supra, is diluted in an appropriatevolume of PBS. Then, 100 μl of the resulting mixture is placed into eachwell of a Reacti-Bind™ metal chelate microtiter plate (Pierce, Rockford,Ill.), incubated at room temperature while shaking, and followed bythree washes with 200 μl each of PBS with 0.05% Tween® 20. The preparedmicrotiter plate can then be used to screen polyclonal antisera for thepresence of BU101 antibodies (see Example 17).

Although pcDNA3. 1/Myc-His is utilized in this example, it is known tothose skilled in the art that other comparable expression systems can beutilized herein with appropriate modifications in reagent and/ortechniques and are within the skill of one of ordinary skill in the art.The largest cloned insert containing the coding region of the BU101 geneis sub-cloned into either (i) a eukaryotic expression vector which maycontain, for example, a cytomegalovirus (CMV) promoter and/or proteinfusible sequences which aid in protein expression and detection, or (ii)a bacterial expression vector containing a superoxide-dismutase (SOD)and CMP-KDO synthetase (CKS) or other protein fusion gene for expressionof the protein sequence. Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in published EPO application No. EP 0 196 056, published Oct.1, 1986, which is incorporated herein by reference, and vectorscontaining fusion sequences of CKS are described in published EPOapplication No. EP 0 331 961, published Sep. 13, 1989, which publicationis also incorporated herein by reference. The purified protein can beused in a variety of techniques, including but not limited to, animalimmunization studies, solid phase immunoassays, etc.

Example 12 Chemical Analysis of Breast Tissue Proteins

A. Analysis of Tryptic Peptide Fragments Using MS. Sera from patientswith breast disease such as breast cancer, sera from patients with nobreast disease, extracts of breast tissues or cells from patients withbreast disease such as breast cancer, extracts of breast tissues orcells from patients with no breast disease, and extracts of tissues orcells from other non-diseased or diseased organs of patients are run ona polyacrylamide gel using standard procedures and stained withCoomassie Blue. Sections of the gel suspected of containing the unknownpolypeptide are excised and subjected to an in-gel reduction,acetamidation and tryptic digestion. P. Jeno et al, Anal. Bio.224:451-455 (1995) and J. Rosenfeld et al, Anal. Bio. 203:173-179(1992). The gel sections are washed with 100 mM NH₄HCO₃ andacetonitrile. The shrunken gel pieces are swollen in digestion buffer(50 mM NH₄HCO₃, 5 mM CaCl₂ and 12.5 μg/ml trypsin) at 4° C. for 45 min.The supernatant is aspirated and replaced with 5 to 10 μl of digestionbuffer without trypsin and allowed to incubate overnight at 37° C.Peptides are extracted with 3 changes of 5% formic acid and acetonitrileand evaporated to dryness. The peptides are adsorbed to approximately0.1 μl of POROS R2 sorbent (Perseptive Biosystems, Framingham, Mass.)trapped in the tip of a drawn gas chromatography capillary tube bydissolving them in 10 μl of 5% formic acid and passing it through thecapillary. The adsorbed peptides are washed with water and eluted with5% formic acid in 60% methanol. The eluant is passed directly into thespraying capillary of an API III mass spectrometer (Perkin-Elmer Sciex,Thornhill, Ontario, Canada) for analysis by nano-electrospray massspectrometry. M. Wilm et al., Int. J. Mass Spectrom. Ion Process136:167-180 (1994) and M. Wilm et al., Anal. Chem. 66:1-8 (1994). Themasses of the tryptic peptides are determined from the mass spectrumobtained off the first quadrupole. Masses corresponding to predictedpeptides can be further analyzed in MS/MS mode to give the amino acidsequence of the peptide.

B. Peptide Fragment Analysis Using LC/MS. The presence of polypeptidespredicted from mRNA sequences found in hyperplastic disease tissues alsocan be confirmed using liquid chromatography/tandem mass spectrometry(LC/MS/MS). D. Hess et al., METHODS, A Companion to Methods inEnzymology 6:227-238 (1994). The serum specimen or tumor extract fromthe patient is denatured with SDS and reduced with dithiothreitol (1.5mg/ml) for 30 min at 90° C. followed by alkylation with iodoacetamide (4mg/mil) for 15 min at 25° C. Following acrylamide electrophoresis, thepolypeptides are electroblotted to a cationic membrane and stained withCoomassie Blue. Following staining, the membranes are washed andsections thought to contain the unknown polypeptides are cut out anddissected into small pieces. The membranes are placed in 500 μlmicrocentrifuge tubes and immersed in 10 to 20 μl of proteolyticdigestion buffer (100 mM Tris-HCl, pH 8.2, containing 0.1 M NaCl, 10%acetonitrile, 2 mM CaCl₂ and 5 μg/ml trypsin) (Sigma, St. Louis, Mo.).After 15 h at 37° C., 3 μl of saturated urea and 1 μl of 100 μg/mltrypsin are added and incubated for an additional 5 h at 37° C. Thedigestion mixture is acidified with 3 μl of 10% trifluoroacetic acid andcentrifuged to separate supernatant from membrane. The supernatant isinjected directly onto a microbore, reverse phase HPLC column and elutedwith a linear gradient of acetonitrile in 0.05% trifluoroacetic acid.The eluate is fed directly into an electrospray mass spectrometer, afterpassing though a stream splitter if necessary to adjust the volume ofmaterial. The data is analyzed following the procedures set forth inExample 12, Section A.

Example 13 Gene Immunization Protocol

A. In Vivo Antigen Expression. Gene immunization circumvents proteinpurification steps by directly expressing an antigen in vivo afterinoculation of the appropriate expression vector. Also, production ofantigen by this method may allow correct protein folding andglycosylation since the protein is produced in mammalian tissue. Themethod utilizes insertion of the gene sequence into a plasmid whichcontains a CMV promoter, expansion and purification of the plasmid andinjection of the plasmid DNA into the muscle tissue of an animal.Preferred animals include mice and rabbits. See, for example, H. Daviset al., Human Molecular Genetics 2:1847-1851 (1993). After one or twobooster immunizations, the animal can then be bled, ascites fluidcollected, or the animal's spleen can be harvested for production ofhybridomas.

B. Plasmid Preparation and Purification. BU101 cDNA insert was releasedfrom the BU101 vector described in Example 11b by digestion with EcoRIand NotI restriction enzymes. The digested plasmid fragments wereelectrophoresed on a 1% Seakem KE agarose/0.5 μg/ml ethidium bromide/TEgel and the bands were visualized by UV illumination. The insertfragment was excised from the gel and purified using the QIAquickprocedure, described supra. The fragment was ligated into an EcoRI/NotIdigested pcDNA3.1 vector (Invitrogen, Carlsbad, Calif.) and transformedinto DH5 alpha cells as described supra. The plasmid DNA was purifiedfrom the bacterial lysate using a QIAprep column. All these techniquesare familiar to one of ordinary skill in the art of molecular biology.

C. Immunization Protocol. Anesthetized animals are immunizedintramuscularly with 0.1-100 μg of the purified plasmid diluted in PBSor other DNA uptake enhancers (Cardiotoxin, 25% sucrose). See, forexample, H. Davis et al, Human Gene Therapy 4:733-740 (1993); and P. W.Wolff et al, Biotechniques 11:474-485 (1991). One to two boosterinjections are given at monthly intervals.

D. Testing and Use of Antiserum. Animals are bled and the resultant seratested for antibody using peptides synthesized from the known genesequence (see Example 16) using techniques known in the art, such aswestern blotting or EIA techniques. Antisera produced by this method canthen be used to detect the presence of the antigen in a patient's tissueor cell extract or in a patient's serum by ELISA or Western blottingtechniques, such as those described in Examples 15 through 18.

Example 14 Production of Antibodies Against BU101

A. Production of Polyclonal Antisera. Antiserum against BU101 wasprepared by injecting rabbits with peptides whose sequences were derivedfrom that of the predicted amino acid sequence of the BU101 consensussequence (SEQUENCE ID NO 3). The synthesis of peptides (SEQ ID NOS16-23) was described in Example 10. Peptides used as immunogen wereeither conjugated to a carrier, keyhole limpet hemocyanin (KLH)(SEQUENCE ID NOS 16-22), prepared as described hereinbelow, orunconjugated (i.e., not conjugated to a carrier such as KLH) (SEQUENCEID NO 21, SEQUENCE ID NO 22, and SEQUENCE ID NO 23).

1. Peptide Conjugation. Peptide was conjugated to maleimide activatedkeyhole limpet hemocyanin (KLH, commercially available as Imject®,available from Pierce Chemical Company, Rockford, Ill.). Imject®contains about 250 moles of reactive maleimide groups per mole ofhemocyanin. The activated KLH was dissolved in phosphate buffered saline(PBS, pH 8.4) at a concentration of about 7.7 mg/ml. The peptide wasconjugated through cysteines occurring in the peptide sequence, or to acysteine previously added to the synthesized peptide in order to providea point of attachment. The peptide was dissolved in dimethyl sulfoxide(DMSO, Sigma Chemical Company, St. Louis, Mo.) and reacted with theactivated KLH at a mole ratio of about 1.5 moles of peptide per mole ofreactive maleimide attached to the KLH. A procedure for the conjugationof peptides (SEQUENCE ID NOS 16-22) is provided hereinbelow. It is knownto the ordinary artisan that the amounts, times and conditions of such aprocedure can be varied to optimize peptide conjugation.

The conjugation reaction described hereinbelow was based on obtaining 3mg of KLH peptide conjugate (“conjugated peptide”), which contains about0.77 μmoles of reactive maleimide groups. This quantity of peptideconjugate usually was adequate for one primary injection and fourbooster injections for production of polyclonal antisera in a rabbit.Briefly, each peptide (SEQUENCE ID NOS 16-22) was dissolved in DMSO at aconcentration of 1.16 μmoles/100 μl of DMSO. One hundred microliters(100 μl) of the DMSO solution were added to 380 μl of the activated KLHsolution prepared as described hereinabove, and 20 μl of PBS (pH 8.4)was added to bring the volume to 500 μl. The reaction was incubatedovernight at room temperature with stirring. The extent of reaction wasdetermined by measuring the amount of unreacted thiol in the reactionmixture. The difference between the starting concentration of thiol andthe final concentration was assumed to be the concentration of peptidewhich has coupled to the activated KLH. The amount of remaining thiolwas measured using Ellman's reagent (5,5′-dithiobis(2-nitrobenzoicacid), Pierce Chemical Company, Rockford, Ill.). Cysteine standards weremade at a concentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35mg of cysteine HCl (Pierce Chemical Company, Rockford, Ill.) in 10 ml ofPBS (pH 7.2) and diluting the stock solution to the desiredconcentration(s). The photometric determination of the concentration ofthiol was accomplished by placing 200 μl of PBS (pH 8.4) in each well ofan Immulon 2® microwell plate (Dynex Technologies, Chantilly, Va.).Next, 10 μl of standard or reaction mixture was added to each well.Finally, 20 μl of Ellman's reagent at a concentration of 1 mg/ml in PBS(pH 8.4) was added to each well. The wells were incubated for 10 minutesat room temperature, and the absorbance of all wells was read at 415 nmwith a microplate reader (such as the BioRad Model 3550, BioRad,Richmond, Calif.). The absorbance of the standards was used to constructa standard curve and the thiol concentration of the reaction mixture wasdetermined from the standard curve. A decrease in the concentration offree thiol was indicative of a successful conjugation reaction. Inaddition, calculation of free thiol in the peptide solution, prior toaddition of the maleimide activated KLH and upon completion of thereaction, allowed determination of the substitution ratio of moles ofpeptide/mole of KLH for each peptide×KLH conjugate. In all cases, thereaction went to completion, and there were approximately 250peptides/KLH molecule for each of the peptide conjugates prepared. Anyunreacted peptide was removed by dialysis against PBS (pH 7.2) at roomtemperature for 6 hours. The conjugate was stored at 2-8° C. if it wasto be used immediately; otherwise, it was stored at −20° C. or colder.

2. Animal Immunization. Female white New Zealand rabbits weighing 2 kgor more were used for raising polyclonal antiserum. Generally, oneanimal was immunized per unconjugated or conjugated peptide(BU101.1-BU101.8, prepared as described hereinabove). One week prior tothe first immunization, 5 to 10 ml of blood were obtained from theanimal to serve as a non-immune prebleed sample.

Each of the unconjugated or conjugated peptides, BUI101.1-BU101.8 (SEQID NOS 16-23), was used to prepare the primary immunogen by emulsifying0.5 ml of the peptide at a concentration of 2 mg/ml in PBS (pH 7.2) with0.5 ml of complete Freund's adjuvant (CFA) (Difco, Detroit, Mich.). Theimmunogen was injected into several sites of the animal viasubcutaneous, intraperitoneal, and/or intramuscular routes ofadministration. Four weeks following the primary immunization, a boosterimmunization was administered. The immunogen used for the boosterimmunization dose was prepared by emulsifying 0.5 ml of the sameunconjugated or conjugated peptide used for the primary immunogen,except that the peptide now was diluted to 1 mg/ml with 0.5 ml ofincomplete Freund's adjuvant (IFA) (Difco, Detroit, Mich.). Again, thebooster dose was administered into several sites and utilizedsubcutaneous, intraperitoneal and intramuscular types of injections. Theanimal was bled (5 ml) two weeks after the booster immunization and theserum was tested for immunoreactivity to the peptide, as describedbelow. The booster and bleed schedule was repeated at 4 week intervalsuntil an adequate titer was obtained. The titer or concentration ofantiserum was determined by microtiter EIA as described in Example 17,below. In addition, apparent affinity values [Kd(app)] were determinedfor some of the antisera (see Example 17). For both titer and apparentaffinity measurements, full length BU101.8 peptide (SEQUENCE ID NO 23)was used as the antigen. An antibody titer of 1:500 or greater wasconsidered an adequate titer for further use and study.

B. Production of Monoclonal Antibody.

1. Immunization Protocol. Mice are immunized using immunogens preparedas described hereinabove, except that the amount of the unconjugated orconjugated peptide for monoclonal antibody production in mice isone-tenth the amount used to produce polyclonal antisera in rabbits.Thus, the primary immunogen consists of 100 μg of unconjugated orconjugated peptide in 0.1 ml of CFA emulsion; while the immunogen usedfor booster immunizations consists of 50 μg of unconjugated orconjugated peptide in 0.1 ml of IFA. Hybridomas for the generation ofmonoclonal antibodies are prepared and screened using standardtechniques. The methods used for monoclonal antibody development followprocedures known in the art such as those detailed in Kohler andMilstein, Nature 256:494 (1975) and reviewed in J. G. R. Hurrel, ed.,Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,Inc., Boca Raton, Fla. (1982). Another method of monoclonal antibodydevelopment which is based on the Kohler and Milstein method is that ofL. T. Mimms et al., Virology 176:604-619 (1990), which is incorporatedherein by reference.

The immunization regimen (per mouse) consists of a primary immunizationwith additional booster immunizations. The primary immunogen used forthe primary immunization consists of 100 μg of unconjugated orconjugated peptide in 50 μl of PBS (pH 7.2) previously emulsified in 50μl of CFA. Booster immunizations performed at approximately two weeksand four weeks post primary immunization consist of 50 μg ofunconjugated or conjugated peptide in 50 μl of PBS (pH 7.2) emulsifiedwith 50 μl IFA. A total of 100 μl of this immunogen is inoculatedintraperitoneally and subcutaneously into each mouse. Individual miceare screened for immune response by microtiter plate enzyme immunoassay(EIA) as described in Example 17 approximately four weeks after thethird immunization. Mice are inoculated either intravenously,intrasplenically or intraperitoneally with 50 μg of unconjugated orconjugated peptide in PBS (pH 7.2) approximately fifteen weeks after thethird immunization.

Three days after this intravenous boost, splenocytes are fused with, forexample, Sp2/0-Agl4 myeloma cells (Milstein Laboratories, England) usingthe polyethylene glycol (PEG) method. The fusions are cultured inIscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal calfserum (FCS), plus 1% hypoxanthine, aminopterin and thymidine (HAT). Bulkcultures are screened by microtiter plate EIA following the protocol inExample 17. Clones reactive with the peptide used an immunogen andnon-reactive with other peptides (i.e., peptides of BU101 not used asthe immunogen) are selected for final expansion. Clones thus selectedare expanded, aliquoted and frozen in IMDM containing 10% FCS and 10%dimethyl-sulfoxide.

2. Production of Ascites Fluid Containing Monoclonal Antibodies.

Frozen hybridoma cells prepared as described hereinabove are thawed andplaced into expansion culture. Viable hybridoma cells are inoculatedintraperitoneally into Pristane treated mice. Ascitic fluid is removedfrom the mice, pooled, filtered through a 0.2μ filter and subjected toan immunoglobulin class G (IgG) analysis to determine the volume of theProtein A column required for the purification.

3. Purification of Monoclonal Antibodies From Ascites Fluid. Briefly,filtered and thawed ascites fluid is mixed with an equal volume ofProtein A sepharose binding buffer (1.5 M glycine, 3.0 M NaCl, pH 8.9)and refiltered through a 0.2μ filter. The volume of the Protein A columnis determined by the quantity of IgG present in the ascites fluid. Theeluate then is dialyzed against PBS (pH 7.2) overnight at 2-8° C. Thedialyzed monoclonal antibody is sterile filtered and dispensed inaliquots. The immunoreactivity of the purified monoclonal antibody isconfirmed by determining its ability to specifically bind to the peptideused as the immunogen by use of the EIA microtiter plate assay procedureof Example 17. The specificity of the purified monoclonal antibody isconfirmed by determining its lack of binding to irrelevant peptides suchas peptides of BU101 not used as the immunogen. The purified anti-BU101monoclonal thus prepared and characterized is placed at either 2-8° C.for short term storage or at −80° C. for long term storage.

4. Further Characterization of Monoclonal Antibody. The isotype andsubtype of the monoclonal antibody produced as described hereinabove canbe determined using commercially available kits (available fromAmersham. Inc., Arlington Heights, Ill.). Stability testing also can beperformed on the monoclonal antibody by placing an aliquot of themonoclonal antibody in continuous storage at 2-8° C. and assayingoptical density (OD) readings throughout the course of a given period oftime.

C. Use of Recombinant Proteins as Immunogens. It is within the scope ofthe present invention that recombinant proteins made as described hereincan be utilized as immunogens in the production of polyclonal andmonoclonal antibodies, with corresponding changes in reagents andtechniques known to those skilled in the art.

Example 15 Purification of Serum Antibodies Which Specifically Bind toBU101 Peptides

Immune sera, obtained as described hereinabove in Examples 13 and/or 14,is affinity purified using immobilized synthetic peptides prepared asdescribed in Example 10, or recombinant proteins prepared as describedin Example 11. An IgG fraction of the antiserum is obtained by passingthe diluted, crude antiserum over a Protein A column (Affi-Gel proteinA, Bio-Rad, Hercules, Calif.). Elution with a buffer (Binding Buffer,supplied by the manufacturer) removes substantially all proteins thatare not immunoglobulins. Elution with 0.1M buffered glycine (pH 3) givesan immunoglobulin preparation that is substantially free of albumin andother serum proteins.

Immunoaffinity chromatography is performed to obtain a preparation witha higher fraction of specific antigen-binding antibody. The peptide usedto raise the antiserum is immobilized on a chromatography resin, and thespecific antibodies directed against its epitopes are adsorbed to theresin. After washing away non-binding components, the specificantibodies are eluted with 0.1 M glycine buffer (pH 2.3). Antibodyfractions are immediately neutralized with 1.0 M Tris buffer (pH 8.0) topreserve immunoreactivity. The chromatography resin chosen depends onthe reactive groups present in the peptide. If the peptide has an aminogroup, a resin such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad,Hercules, Calif.). If coupling through a carboxy group on the peptide isdesired, Affi-Gel 102 can be used (Bio-Rad, Hercules, Calif.). If thepeptide has a free sulfhydryl group, an organomercurial resin such asAffi-Gel 501 (Bio-Rad, Hercules, Calif.) or SulkfoLink™ (Pierce,Rockford, Ill.) can be used. The amount of peptide immobilized on theresin can be determined using Nano Orange™ (Molecular Probes, Eugene,Oreg.).

Alternatively, spleens can be harvested and used in the production ofhybridomas to produce monoclonal antibodies following routine methodsknown in the art as described hereinabove.

Example 16 Western Blotting of Tissue Samples

Protein extracts were prepared by homogenizing tissue samples in 0.1MTris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, 1.0 mM1,4-dithiothreitol, 10 μg/ml leupeptin and 1.0 mMphenylmethylsulfonylfluoride (Kain et al., Biotechniques 17:982 (1994)).Following homogenization, the homogenates were centrifuged at 4° C. for5 minutes to separate supernate from debris. For protein quantitation,3-10 μL of supernate was added to 1.5 ml of bicinchoninic acid reagent(Sigma, St. Louis, Mo.), and the resulting absorbance at 562 nm wasmeasured.

For SDS-PAGE, samples were adjusted to desired protein concentrationwith Tricine Buffer (Novex, San Diego, Calif.), mixed with an equalvolume of 2×Tricine sample buffer (Novex, San Diego, Calif.), and heatedfor 5 minutes at 100° C. in a thermal cycler. Samples were then appliedto a Novex 10-20% Precast Tricine Gel for electrophoresis. Followingelectrophoresis samples were transferred from the gels to nitrocellulosemembranes in Novex Tris-Glycine Transfer buffer. Membranes were thenprobed with specific anti-peptide antibodies using the reagents andprocedures provided in the Western Lights Plus or Western Lights(Tropix, Bedford, Mass.) chemiluminesence detection kits.Chemiluminesent bands were visualized by exposing the developedmembranes to Hyperfilm ECL (Amersham, Arlington Heights, Ill.).

FIG. 5 shows the results of the western blot performed on a panel oftissue protein extracts (CloneTech, Palo Alto, Calif.) using BU101.8antiserum (see Example 14). Each lane of FIG. 5 represents a differenttissue protein extract (1, stomach; 2, blank; 3, heart; 4, placenta; 5,spleen; 6, brain; 7, kidney; 8, breast tumor; 9, lung;, 10 liver; 11ovary; 12, markers). A band at 6.5 kD (arrow), as determined by proteinsize markers (lane 12), was detected only in the breast tissue extract(lane 8) and not in any other of the tissue extracts. In other westernblots (data not shown), a 6.5 kD band was also observed in 5 of 9 breastcancer tissue protein extracts and 1 of 6 normal breast tissue proteinextracts.

Competition experiments were carried out in an analogous manner asabove, with the following exception; the primary antibodies(anti-peptide polyclonal antisera) were pre-incubated for 30 minutes atroom temperature with varying concentrations of peptide immunogen priorto exposure to the nitrocellulose filter. Development of the Western wascontinued as above. Antibody binding to the band at 6.5 kD was inhibitedat a concentration of 100 nM of BU101.3 peptide.

After visualization of the bands on film, the bands can also bevisualized directly on the membranes by the addition and development ofa chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate(BCIP). This chromogenic solution contains 0.016% BCIP in a solutioncontaining 100 mM NaCl, 5 mM MgCl₂ and 100 mM Tris-HCl, pH 9.5. Thefilter is incubated in the solution at room temperature until the bandsdevelop to the desired intensity. Molecular mass determination is madebased upon the mobility of pre-stained molecular weight standards(Novex, San Diego, Calif.) or biotinylated molecular weight standards(Tropix, Bedford, Mass.).

Example 17 EIA Microtiter Plate Assay

The immunoreactivity of antiserum obtained from rabbits, as described inExample 14, was determined by means of a microtiter plate EIA (Table 2).Briefly, synthetic peptides, BU101.1-BU101.8, prepared as described inExample 10, were dissolved in carbonate buffer (50 mM, pH 9.6) to afinal concentration of 2 mg/ml. Next, 100 μl of the peptide or proteinsolution was placed in each well of an Immulon 2® microtiter plate(Dynex Technologies, Chantilly, Va.). The plate was incubated overnightat room temperature and then washed four times with deionized water. Thewells were blocked by adding 125 μl of Superblock® (Pierce ChemicalCompany, Rockford, Ill.) to each well and then immediately discardingthe solution. This blocking procedure was performed three times.Antiserum obtained from immunized rabbits prepared as previouslydescribed was diluted in a protein blocking agent (e.g., a 3%Superblock® solution) in PBS containing 0.05% Tween-20® (monolauratepolyoxyethylene ether) (Sigma Chemical Company, St. Louis, Mo.) and0.05% sodium azide at dilutions of 1:500, 1:2500, 1:12,500, 1:62,500 and1:312,500 and placed in each well of the coated microtiter plate. Thewells then were incubated for three hours at room temperature. Each wellwas washed four times with deionized water. One hundred microliters (100μl) of alkaline phosphatase-conjugated goat anti-rabbit IgG (SouthernBiotech, Birmingham, Ala.), diluted 1:2000 in 3% Superblock® solution inphosphate buffered saline containing 0.05% Tween 20® and 0.05% sodiumazide, was added to each well. The wells were incubated for two hours atroom temperature. Next, each well was washed four times with deionizedwater. One hundred microliters (100 μl) of paranitrophenyl phosphatesubstrate (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) thenwas added to each well. The wells were incubated for thirty minutes atroom temperature. The absorbance at 405 nm was read in each well.Positive reactions were identified by an increase in absorbance at 405nm in the test well above that absorbance given by a non-immune serum(negative control). A positive reaction was indicative of the presenceof detectable anti-BU101 antibodies. Titers of the anti-peptide antiserawere calculated from the previously described dilutions of antisera anddefined as the calculated dilution where A_(405nm)=0.5 OD.

In addition to titers, apparent affinities [Kd(app)] were alsodetermined for some of the anti-peptide antisera (Table 2). EIAmicrotiter plate assay results were used to derive the apparentdissociation constants (Kd) based on an analog of the Michaelis-Mentenequation (V. Van Heyningen, Methods in Enzymology, Vol.121, p. 472(1986) and further described in X. Qiu, et al, Journal of Immunology,Vol. 156, p. 3350 (1996)):$\left\lbrack {{Ag} - {Ab}} \right\rbrack = {{\left\lbrack {{Ag} - {Ab}} \right\rbrack_{\max} \times \frac{\lbrack{Ab}\rbrack}{\lbrack{Ab}\rbrack = {Kd}}}\quad -}$

where [Ag-Ab] is the antigen-antibody complex concentration,[Ag-Ab]_(max) is the maximum complex concentration, [Ab] is the antibodyconcentration, and K_(d) is the dissociation constant. During the curvefitting, the [Ag-Ab] was replaced with the background subtracted valueof the OD_(405nm) at the given concentration of Ab. Both K_(d) and[OD_(405nm)]_(max), which corresponds to the [Ag-Ab]_(max), were treatedas fitted parameters. The software program Origin was used for the curvefitting.

TABLE 2 Titer and Kd(app) of polyclonal antibodies produced againstBU101 Peptide Immunogen Peptide Conjugated? 13 week Titer Kd(app)BU101.1 yes/KLH 12,000 10^(−7.3) BU101.2 yes/KLH 10,000 10^(−7.7)BU101.3 yes/KLH 54,000 10^(−7.8) BU101.4 yes/KLH 8900 10^(−7.0) BU101.5yes/KLH <500 10^(−7.3) BU101.6 no 12,500 10^(−7.5) BU101.6 yes/KLH 470010^(−7.5) BU101.7 no 51,000 10^(−7.8) BU101.7 yes/KLH 43,000 10^(−7.8)BU101.8 no 47,000 10^(−7.7)

Example 18 Coating of Solid Phase Particles

A. Coating of Microparticles with Antibodies Which Specifically Bind toBU101 Antigen. Affinity purified antibodies which specifically bind toBU101 protein (see Example 15) are coated onto microparticles ofpolystyrene, carboxylated polystyrene, polymethylacrylate or similarparticles having a radius in the range of about 0.1 to 20 μm.Microparticles may be either passively or actively coated. One coatingmethod comprises coating EDAC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (AldrichChemical Co., Milwaukee, Wis.) activated carboxylated latexmicroparticles with antibodies which specifically bind to BU101 protein,as follows. Briefly, a final 0.375% solid suspension of resin washedcarboxylated latex microparticles (available from Bangs Laboratories,Carmel, Ind. or Serodyn, Indianapolis, Ind.) are mixed in a solutioncontaining 50 mM MES buffer, pH 4.0 and 150 mg/l of affinity purifiedanti-BU101 antibody (see Example 14) for 15 min in an appropriatecontainer. EDAC coupling agent is added to a final concentration of 5.5μg/ml to the mixture and mixed for 2.5 h at room temperature.

The microparticles then are washed with 8 volumes of a Tween 20®/sodiumphosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2μm Microgon Filtration module. Washed microparticles are stored in anappropriate buffer which usually contains a dilute surfactant andirrelevant protein as a blocking agent, until needed.

B. Coating of ¼ Inch Beads. Antibodies which specifically bind toBU101-antigen also may be coated on the surface of ¼ inch polystyrenebeads by routine methods known in the art (Snitman et al, U.S. Pat. No.5,273,882, incorporated herein by reference) and used in competitivebinding or EIA sandwich assays.

Polystyrene beads first are cleaned by ultrasonicating them for about 15seconds in 10 mM NaHCO₃ buffer at pH 8.0. The beads then are washed indeionized water until all fines are removed. Beads then are immersed inan antibody solution in 10 mM carbonate buffer, pH 8 to 9.5. Theantibody solution can be as dilute as 1 μg/ml in the case of highaffinity monoclonal antibodies or as concentrated as about 500 μg/ml forpolyclonal antibodies which have not been affinity purified. Beads arecoated for at least 12 hours at room temperature, and then they arewashed with deionized water. Beads may be air dried or stored wet (inPBS, pH 7.4). They also may be overcoated with protein stabilizers (suchas sucrose) or protein blocking agents used as non-specific bindingblockers (such as irrelevant proteins, Carnation skim milk, Superblock®,or the like).

Example 19 Microparticle Enzyme Immunoassay (MEIA)

BU101 antigens are detected in patient test samples by performing astandard antigen competition EIA or antibody sandwich EIA and utilizinga solid phase such as microparticles (MEIA). The assay can be performedon an automated analyzer such as the IMx® Analyzer (Abbott Laboratories,Abbott Park, Ill.).

A. Antibody Sandwich EIA. Briefly, samples suspected of containing BU101antigen are incubated in the presence of anti-BU101 antibody-coatedmicroparticles (prepared as described in Example 17) in order to formantigen/antibody complexes. The microparticles then are washed and anindicator reagent comprising an antibody conjugated to a signalgenerating compound (i.e., enzymes such as alkaline phosphatase orhorseradish peroxide) is added to the antigen/antibody complexes or themicroparticles and incubated. The microparticles are washed and thebound antibody/antigen/antibody complexes are detected by adding asubstrate (e.g., 4-methyl umbelliferyl phosphate (MUP), or OPD/peroxide,respectively), that reacts with the signal generating compound togenerate a measurable signal. An elevated signal in the test sample,compared to the signal generated by a negative control, detects thepresence of BU101 antigen. The presence of BU101 antigen in the testsample is indicative of a diagnosis of a breast disease or condition,such as breast cancer.

B. Competitive Binding Assay. The competitive binding assay uses apeptide or protein that generates a measurable signal when the labeledpeptide is contacted with an anti-peptide antibody coated microparticle.This assay can be performed on the IMx® Analyzer (available from AbbottLaboratories, Abbott Park, Ill.). The labeled peptide is added to theBU101 antibody-coated microparticles (prepared as described in Example17) in the presence of a test sample suspected of containing BU101antigen, and incubated for a time and under conditions sufficient toform labeled BU101 peptide (or labeled protein)/bound antibody complexesand/or patient BU101 antigen/bound antibody complexes. The BU101 antigenin the test sample competes with the labeled BU101 peptide (or BU101protein) for binding sites on the microparticle. BU101 antigen in thetest sample results in a lowered binding of labeled peptide and antibodycoated microparticles in the assay since antigen in the test sample andthe BU101 peptide or BU101 protein compete for antibody binding sites. Alowered signal (compared to a control) indicates the presence of BU101antigen in the test sample. The presence of BU101 antigen suggests thediagnosis of a breast disease or condition, such as breast cancer.

The BU101 polynucleotides and the proteins encoded thereby which areprovided and discussed hereinabove are useful as markers of breasttissue disease, especially breast cancer. Tests based upon theappearance of this marker in a test sample such as blood, plasma orserum can provide low cost, non-invasive, diagnostic information to aidthe physician to make a diagnosis of cancer, to help select a therapyprotocol, or to monitor the success of a chosen therapy. This marker mayappear in readily accessible body fluids such as blood, urine or stoolas antigens derived from the diseased tissue which are detectable byimmunological methods. This marker may be elevated in a disease state,altered in a disease state, or be a normal protein of the breast whichappears in an inappropriate body compartment.

25 237 base pairs nucleic acid single linear unknown 1 TCCAAATCACTCATTGTTTG TGAAAGCTGA GCTCACAGCA AAACAAGCCA CCATGAAGCT 60 GTCGGTGTGTCTCCTGCTGG TCACGCTGGC CCTCTGCTGC TACCAGGCCA ATGCCGAGTT 120 CTGCCCAGCTCTTGTTTCTG AGCTGTTAGA CTTCTTCTTC ATTAGTGAAC CTCTGTTCAA 180 GTTAAGTCTTGCCAAATTTG ATGCCCCTCC GGAAGCTGTT GCAGCCAAGT TAGGAGT 237 230 base pairsnucleic acid single linear unknown 2 CCGGAAGCTG TTGCAGCCAA GTTAGGAGTGAAGAGATGCA CGGATCAGAT GTCCCTTCAG 60 AAACGAAGCC TCATTGCGGA AGTCCTGGTGAAAATATTGA AGAAATGTAG TGTGTGACAT 120 GTAAAAACTT TCATCCTGGT TTCCACTGTCTTTCAATGAC ACCCTGATCT TCACTGCAGA 180 ATGTAAAGGT TTCAACGTCT TGCTTTAATAAATCACTTGC TCTCCACGTC 230 494 base pairs nucleic acid single linearunknown 3 CTCCCTAGGT ACAAATAGCC CTGGGCTCTG CAGCTCCACA GGCTCCTGGGGTGGAGTCCA 60 AATCACTCAT TGTTTGTGAA AGCTGAGCTC ACAGCAAAAC AAGCCACCATGAAGCTGTCG 120 GTGTGTCTCC TGCTGGTCAC GCTGGCCCTC TGCTGCTACC AGGCCAATGCCGAGTTCTGC 180 CCAGCTCTTG TTTCTGAGCT GTTAGACTTC TTCTTCATTA GTGAACCTCTGTTCAAGTTA 240 AGTCTTGCCA AATTTGATGC CCCTCCGGAA GCTGTTGCAG CCAAGTTAGGAGTGAAGAGA 300 TGCACGGATC AGATGTCCCT TCAGAAACGA AGCCTCATTG CGGAAGTCCTGGTGAAAATA 360 TTGAAGAAAT GTAGTGTGTG ACATGTAAAA ACTTTCATCC TGGTTTCCACTGTCTTTCAA 420 TGACACCCTG ATCTTCACTG CAGAATGTAA AGGTTTCAAC GTCTTGCTTTAATAAATCAC 480 TTGCTCTCCA CGTC 494 482 base pairs nucleic acid singlelinear unknown 4 CCACGCGTCC GCCCACGCGT CCGTCCAAAT CACTCATTGT TTGTGAAAGCTGAGCTCACA 60 GCAAAACAAG CCACCATGAA GCTGTCGGTG TGTCTCCTGC TGGTCACGCTGGCCCTCTGC 120 TGCTACCAGG CCAATGCCGA GTTCTGCCCA GCTCTTGTTT CTGAGCTGTTAGACTTCTTC 180 TTCATTAGTG AACCTCTGTT CAAGTTAAGT CTTGCCAAAT TTGATGCCCCTCCGGAAGCT 240 GTTGCAGCCA AGTTAGGAGT GAAGAGATGC ACGGATCAGA TGTCCCTTCAGAAACGAAGC 300 CTCATTGCGG AAGTCCTGGT GAAAATATTG AAGAAATGTA GTGTGTGACATGTAAAAACT 360 TTCATCCTGG TTTCCACTGT CTTTCAATGA CACCCTGATC TTCACTGCAGAATGTAAAGG 420 TTTCAACGTC TTGCTTTAAT AAATCACTTG CTCTCCACGT AAAAAAAAAAAAAAAAAAAA 480 GG 482 68 base pairs nucleic acid single linear unknown 5AGCTCGGAAT TCCGAGCTTG GATCCTCTAG AGCGGCCGCC GACTAGTGAG CTCGTCGACC 60CGGGAATT 68 68 base pairs nucleic acid single linear unknown 6AATTAATTCC CGGGTCGACG AGCTCACTAG TCGGCGGCCG CTCTAGAGGA TCCAAGCTCG 60GAATTCCG 68 24 base pairs nucleic acid single linear unknown 7AGCGGATAAC AATTTCACAC AGGA 24 18 base pairs nucleic acid single linearunknown 8 TGTAAAACGA CGGCCAGT 18 19 base pairs nucleic acid singlelinear unknown 9 ACTTGAACAG AGGTTCACT 19 20 base pairs nucleic acidsingle linear unknown 10 CAGCCAAGTT AGGAGTTGAA 20 19 base pairs nucleicacid single linear unknown 11 GAGATGCACG GATCAGATG 19 20 base pairsnucleic acid single linear unknown 12 TTTTACGTGG AGAGCAAGTG 20 20 basepairs nucleic acid single linear unknown 13 TAATACGACT CACTATAGGG 20 30base pairs nucleic acid single linear unknown 14 GCGGCCGCCC ACACTACATTTCTTCAATAT 30 90 amino acids amino acid single linear None unknown 15Met Lys Leu Ser Val Cys Leu Leu Leu Val Thr Leu Ala Leu Cys Cys 1 5 1015 Tyr Gln Ala Asn Ala Glu Phe Cys Pro Ala Leu Val Ser Glu Leu Leu 20 2530 Asp Phe Phe Phe Ile Ser Glu Pro Leu Phe Lys Leu Ser Leu Ala Lys 35 4045 Phe Asp Ala Pro Pro Glu Ala Val Ala Ala Lys Leu Gly Val Lys Arg 50 5560 Cys Thr Asp Gln Met Ser Leu Gln Lys Arg Ser Leu Ile Ala Glu Val 65 7075 80 Leu Val Lys Ile Leu Lys Lys Cys Ser Val 85 90 15 amino acids aminoacid single linear None unknown 16 Glu Phe Cys Pro Ala Leu Val Ser GluLeu Leu Asp Phe Phe Phe 1 5 10 15 16 amino acids amino acid singlelinear None unknown 17 Ile Ser Glu Pro Leu Phe Lys Leu Ser Leu Ala LysPhe Asp Ala Cys 1 5 10 15 16 amino acids amino acid single linear Noneunknown 18 Ser Leu Ala Lys Phe Asp Ala Pro Pro Glu Ala Val Ala Ala LysCys 1 5 10 15 15 amino acids amino acid single linear None unknown 19Glu Ala Val Ala Ala Lys Leu Gly Val Lys Arg Cys Thr Asp Gln 1 5 10 15 16amino acids amino acid single linear None unknown 20 Met Ser Leu Gln LysArg Ser Leu Ile Ala Glu Val Leu Val Lys Cys 1 5 10 15 22 amino acidsamino acid single linear None unknown 21 Met Ser Leu Gln Lys Arg Ser LeuIle Ala Glu Val Leu Val Lys Ile 1 5 10 15 Leu Lys Lys Cys Ser Val 20 45amino acids amino acid single linear None unknown 22 Leu Ala Lys Phe AspAla Pro Pro Glu Ala Val Ala Ala Lys Leu Gly 1 5 10 15 Val Lys Arg CysThr Asp Gln Met Ser Leu Gln Lys Arg Ser Leu Ile 20 25 30 Ala Glu Val LeuVal Lys Ile Leu Lys Lys Cys Ser Val 35 40 45 69 amino acids amino acidsingle linear None unknown 23 Glu Phe Cys Pro Ala Leu Val Ser Glu LeuLeu Asp Phe Phe Phe Ile 1 5 10 15 Ser Glu Pro Leu Phe Lys Leu Ser LeuAla Lys Phe Asp Ala Pro Pro 20 25 30 Glu Ala Val Ala Ala Lys Leu Gly ValLys Arg Cys Thr Asp Gln Met 35 40 45 Ser Leu Gln Lys Arg Ser Leu Ile AlaGlu Val Leu Val Lys Ile Leu 50 55 60 Lys Lys Cys Ser Val 65 8 aminoacids amino acid single linear unknown 24 Asp Tyr Lys Asp Asp Asp AspLys 1 5 21 amino acids amino acid single linear unknown 25 Glu Gln LysLeu Ile Ser Glu Glu Asp Leu Asn Met His Thr Glu His 1 5 10 15 His HisHis His His 20

We claim:
 1. A method of detecting the presence of a target BU101polynucleotide in a test sample, comprising: (a) contacting said testsample with at least one BU101-specific polynucleotide or exactcomplement thereof, (b) detecting the presence of said target BU101polynucleotide in the sample, wherein said BU101-specific polynucleotideconsists of a nucleic acid sequence selected from the group consistingof (I) SEQUENCE ID NO: 1; (ii) SEQUENCE ID NO: 3; iii) SEQUENCE ID NO:4; and (iv) exact complements of (I), (ii), or (iii).
 2. The method ofclaim 1, wherein said target BU101 polynucleotide is attached to a solidphase prior to performing step (a).
 3. A method of detecting mRNA ofBU101 in a test sample, comprising: (a) performing reverse transcriptionon the test sample with at least one BU101 oligonucleotide primer inorder to produce cDNA; (b) amplifying the cDNA obtained from step (a)using BU101 oligonucleotides as sense and antisense primers to obtainBU101 amplicon; and (c) detecting the presence of said BU101 amplicon inthe test sample, wherein said BU101 oligonucleotides utilized in steps(a) and (b) consist of nucleic acid sequences selected from the groupconsisting of (I) SEQUENCE ID NO: 1; (ii) SEQUENCE ID NO: 3; (iii)SEQUENCE ID NO: 4; and (iv) exact complements of (I), (ii) or (iii). 4.The method of claim 3, wherein said test sample is reacted with a solidphase prior to performing one of steps (a), (b), or (c).
 5. The methodof claim 3, wherein said detection step comprises utilizing a detectablelabel capable of generating a measurable signal.
 6. A method ofdetecting a target BU101 polynucleotide in a test sample suspected ofcontaining said target, comprising: (a) contacting said test sample withat least one BU101 oligonucleotide as a sense primer and with at leastone BU101 oligonucleotide as an anti-sense primer and amplifying toobtain a first stage reaction product; (b) contacting said first stagereaction product with at least one other BU101 oligonucleotide to obtaina second stage reaction product; (c) detecting said second stagereaction product as an indication of the presence of the target BU101polynucleotide, wherein the BU101 oligonucleotides utilized in steps (a)and (b) consist of nucleic acid sequences selected from the groupconsisting of (I) SEQUENCE ID NO: 1; (ii) SEQUENCE ID NO: 3; (iii)SEQUENCE ID NO: 4; and (iv) exact complements of (I), (ii) or (iii). 7.The method of claim 6, wherein said test sample is reacted with a solidphase prior to performing one of steps (a), (b), or (c).
 8. The methodof claim 6, wherein said detection step comprises utilizing a detectablelabel capable of generating a measurable signal.
 9. The method of claim8, wherein said detectable label is reacted to a solid phase.
 10. A testkit useful for detecting BU101 polynucleotide in a test sample,comprising a container containing at least one BU101 polynucleotide,wherein said polynucleotide consists of a nucleic acid selected from thegroup consisting of (I) SEQUENCE ID NO: 1; (ii) SEQUENCE ID NO: 3; (iii)SEQUENCE ID NO: 4; and (v) (iV) exact complements of (I), (ii) or (iii).11. A purified polynucleotide, wherein said polynucleotide consists of anucleic acid selected from the group consisting of (I) SEQUENCE ID NO:1; (ii) SEQUENCE ID NO: 3; (iii) SEQUENCE ID NO: 4; and (iv) exactcomplements of (I), (ii), or (iii).
 12. A vector comprising an insertconsisting of a nucleic acid sequence derived from BU101, wherein saidnucleic acid sequence is operably linked to a control sequencecompatible with a desired host, and wherein said nucleic acid sequenceconsists of a nucleic acid (is) selected from the group consisting of(I) SEQUENCE ID NO: 1; (ii) SEQUENCE ID NO: 3; (iii) SEQUENCE ID NO: 4;and (iv) exact complements of (I), (ii), or (iii).
 13. A celltransfected with the vector of claim
 12. 14. A cell transfected with anucleic acid sequence, wherein said nucleic acid sequence consists of asequence selected from the group consisting of SEQUENCE ID NO: 1,SEQUENCE ID NO: 3, SEQUENCE ID NO: 4, and exact complements thereof. 15.A composition of matter comprising an isolated BU101 polynucleotide,wherein said polynucleotide consists of a nucleic acid molecule (is)selected from the group consisting of (I) SEQUENCE ID NO: 1; (ii)SEQUENCE ID NO: 3; (iii) SEQUENCE ID NO: 4; and (iv) exact complementsof (I), (ii), or (iii).
 16. The test kit of claim 10 further comprisinga container with tools useful for collection of said sample, wherein thetools are selected from the group consisting of lancets, absorbentpaper, cloth, swabs and cups.
 17. An isolated polynucleotide consistingof a nucleic acid sequence represented by SEQUENCE ID NO:
 4. 18. Themethod of claim 1, wherein said method is useful for detecting diseasesof the breast.
 19. The method of claim 3, wherein said method is usefulfor detecting diseases of the breast.
 20. The method of claim 6, whereinsaid method is useful for detecting diseases of the breast.
 21. Anoligonucleotide probe or primer consisting of approximately at leastabout 10 nucleotides of a sequence selected from the group consisting ofSEQUENCE ID NO: 1, SEQUENCE ID NO: 3 and SEQUENCE ID NO:
 4. 22. Anoligonucleotide probe or primer consisting of approximately at leastabout 15 nucleotides of a sequence selected from the group consisting ofSEQUENCE ID NO: 1, SEQUENCE ID NO: 3 and SEQUENCE ID NO: 4.